|Role of Phospholipid Metabolism and G Protein in the Action Induced by
Clostridium Perfringens Alpha-Toxin
|Masahiro Nagahama*1, Masataka Oda1, Sadayuki Ochi2, Keiko Kobayashi1 and Jun Sakurai1
|1Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
|2Department of Microbiology, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Department of Microbiology
Faculty of Pharmaceutical Sciences
Tokushima Bunri University, Yamashiro-cho
Tokushima 770-8514, Japan
|Received February 06, 2012; Accepted March 20, 2012; Published March 23,
|Citation: Nagahama M, Oda M, Ochi S, Kobayashi K, Sakurai J (2012) Role
of Phospholipid Metabolism and G Protein in the Action Induced by Clostridium
Perfringens Alpha-Toxin. J Glycom Lipidom S3:001. doi:10.4172/2153-0637.S3-001
|Copyright: © 2012 Nagahama M, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
|Alpha-toxin (370 residues) of Clostridium perfringens is the key virulence determinant in gas gangrene and has also
been implicated in the pathogenesis of sudden death syndrome in young animals. Alpha-toxin possesses phospholipase
C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis and lethality. The structure of the toxin
reveals two domains: the N-terminal domain containing the catalytic active site and the C-terminal domain involving the
binding to membranes. Recent research data showed that alpha-toxin-induced biological activities are responsible for
the activation of phospholipid metabolism via a pertussis toxin (PT)-sensitive GTP-binding protein, Gi. In this review, we
summary the role of phospholipid metabolism and G protein in the biological activities induced by alpha-toxin. Discussed
are activations of the arachidonic acid cascade (Section 1), the phospholipid metabolism (Section 2), the sphingomeylin
metabolism (Section 3) and TrkA signaling (Section 4) induced by alpha-toxin.
|C. perfringens alpha-toxin; Phospholipase C; Sphingomyelinase;
|AA: Arachidonic Acid; CF: Caboxyfluorescein;
DAG: Diacylglycerol; PA: Phosphatidic Acid; PC: Phosphatidyl
Choline; PI: Phosphatidyl Inositol; PLC: Phospholipase C; PT: Pertusis-
Toxin; S1P: Sphingosine 1-Phosphate; SMase: Sphingomyelinase; Tm:
Phase Transition temperature; TXA2: Thromboxane A2
|Clostridium perfringens produces alpha-toxin, which is an
important virulence factor in gas gangrene [1-3]. Alpha-toxin
is hemolytic, dermonecrotic, and lethal. Furthermore, it has
phospholipase C (PLC) and sphingomyelinase (SMase) activities [1-
3]. The toxin has been shown to damage the membranes of various
mammalian cells [1-3] as well as artificial membranes . The structure
of alpha-toxin shows two domains, the N-domain (residues 1-250)
contains the catalytic active site and the C-domain (residues 251-370)
responsible for the binding to membranes (Figure 1) . The gene
encoding alpha-toxin , Bacillus cereus PLC (BCPLC) , and PLCs
from C. bifermentans  and Listeria monocytogenes  have been
isolated and their nucleotide sequences were determined. The results
show that the deduced amino acid sequences of alpha-toxin and these
enzymes exhibit significant homology up to approximately 250 residues
from the N-terminus. From these findings, alpha-toxin was found to
belong to the PLC family. The C-domain is similar to the C2 domain
of intracellular eukaryotic proteins involved in vesicular transport and
signal transduction [10,11].
||Figure 1: Structure of alpha-toxin.
|We reported that alpha-toxin has two tightly bound zinc metals and
an exchangeable divalent cation: residues His-68, -126 and -136 bind
an exchangeable divalent cation required for binding to membranes,
His-148 and Glu-152 bind one zinc ion essential for the active site
of the toxin, and His-11 and Asp-130 tightly bind the other zinc ion
required for maintenance of the structure [12,13]. We also reported
that Tyr-57 and –65 plays a role in the penetration of the toxin into the
bilayer of membranes and access of the catalytic site to sphingomyelin
in membranes, but do not participate in the enzymatic activity (Figure
|Alpha-toxin induced the leakage of caboxyfluorescein (CF) from
liposomes composed of cholesterol and phosphatidylcholine (PC)
containing unsaturated fatty acyl residues or shorter chains of saturated fatty acyl residues (12 and 14 carbon atoms), and the toxin-induced
release of CF decreased as the chain of the acylresidues of PC increased
in length . Therefore, it is possible that the membrane-damaging
effect of alpha-toxin on liposomes is related to the phase transition
temperature (Tm) of PC in PC-cholesterol liposomes . We prepared
various PCs (C18:0/C18:1) with an unsaturated bond in the sn-2 acyl
chain. Differential scanning calorimetry showed that Tm was minimal
when the triple bond was positioned at C (9) in the sn-2 acyl chain.
Our result shows that the binding to liposomes of the toxin and the
alpha-toxin-induced release of CF from liposomes increased with a
decrease in the Tm of the PC in liposomes, suggesting that an increase
in membrane fluidity promotes the binding of the toxin to liposomes . It appears that an increase in membrane fluidity results in the
toxin being inserted into the bilayer of the membrane. Accordingly,
it is concluded that the membrane-damaging action of alpha-toxin is
closely related to the membrane fluidity of liposomes.
|We have reported that the alpha-toxin-induced contraction of
isolated rat aorta and ileum [16-18], and hemolysis of rabbit erythrocytes
[19-21] are dependent on the activation of glycerophospholipid
metabolism. We have also demonstrated that the alpha-toxin-induced
hemolysis of sheep erythrocytes is dependent on the activation of
the sphingomyelin metabolic system through GTP-binding proteins,
especially the formation of sphingosine 1-phosphate [22,23].Therefore,
the toxin-induced activation of phospholipid metabolism through G
protein is closely involved in the biological activities. In the present
review, we demonstrate the relationship between phospholipid
metabolism via G protein and the biological activities induced by
|Activation of the arachidonic acid cascade by alpha-toxin
|Alpha-toxin potentiated contractions of the isolated rat vas deferens
elicited by noradrenaline , and caused a rise in blood pressure with
a decrease in blood flow . The toxin caused contractions of the
isolated rat aorta in a dose-dependent manner . The response to
repeated doses of the toxin was found to be tachyphylactic. The toxininduced
contractions were not inhibited by an alpha-adrenoreceptor
blocking agent (phentolamine), a histamine receptor blocking
agent (chlorpheniramine), or a muscarinic receptor blocking agent
(atropine). The response to the toxin therefore seems to be the result
of a direct effect on the aorta. Alpha-toxin-induced contractions were
inhibited by Ca channel blockers such as verapamil and nifedipine
. Furthermore, the toxin-induced contractionswere not observed
in Ca-free medium. The data indicate that the direct action of the
toxin is mainly due to an increase in Ca permeability across the
smooth muscle cell membranes . Labelling by 32P phosphate of
phosphatidylinositol (PI) and phosphatidic acid (PA) in fragments
of the aorta was enhanced by alpha-toxin . Therefore, Ca influx
caused by the toxin is thought to be associated with the stimulation
of phospholipid metabolism. On the other hand, it is possible that the
increased turnover of phospholipids caused by the toxin stimulates the
breakdown of PI into diacylglycerol (DAG) and inositolphosphates.
Alpha-toxin-induced contractions were significantly inhibited by
cyclo-oxygenase inhibitors such as indomethacin and aspirin .
Furthermore, the toxin was found to stimulate release of arachidonic
acid (AA), indicating that stimulation of AA release relates to activation
of phospholipid metabolism by the toxin. The release of AA caused by
the toxin was greater in the presence than absence of indomethacin,
suggesting that AA is mainly metabolized to cyclo-oxygenase products,
indicating the toxin-induced contraction to be associated with cyclooxygenase
product(s) metabolized from AA . The toxin-evoked
contraction and AA release were unaffected by the phospholipase A2
inhibitor quinacrine. Therefore, the release of AA seems to be due to the
hydrolysis of diacylglycerol by diacylglycerol lipase. The thromboxane
synthetase inhibitor OKY-046 and thromboxane A2 (TXA2) antagonist
ONO-3708 were found to inhibit contractions of the aorta produced by
the toxin. OKY-046 and indomethacin blocked the production of TXA2
caused by the toxin. TXA2 is produced enzymatically by endothelial
cells of rabbit, bovine and human umbilical blood vessels . The
toxin did not induce contractions after treatment of the isolated aorta
with collagenase or rubbing of the tissue to remove endothelial cells
from the intimal surface of the tissue . Thus, the toxin-induced
contractions of the isolated rat aorta associated with the production
of TXA2 required an intact endothelium. Furthermore, TMB-8 (intracellular Ca2+ antagonist), trifluoperazine, W-7(calmodulin
inhibitor) and H-7 (PKC inhibitor) significantly blocked the toxininduced
contractions . From these observations, in endothelial
cells of the rat aorta, alpha-toxin stimulates phosphotidylinositol
metabolism and arachidonic acid release, leading to the production of
TXA2 and the phosphorylation of myosin light chain which then elicits
contractions of the adjacent aorta smooth muscle (Figure 2).
||Figure 2: Mechanism of aortic contraction induced by alpha-toxin.
|Activation of phosholipid metabolism through GTP-binding
protein induced by alpha-toxin in rabbit erythrocytes
|Alpha-toxin induces hot-cold hemolysis of rabbit erythrocytes.
The toxin induced production of PA in a dose-dependent manner
when incubated with erythrocytes at 37°C. We have reported that
incubation of rabbit erythrocyte membranes with the toxin results
in biphasic production of PA  (Figure 3). When erythrocyte
membranes are incubated with the toxin in the presence of [γ32P]ATP
at 37°C, the formation of [32P] phosphatidic acid (PA) is biphasic, the
first phase lasting 30s and the second phase, c. 20 min . Treatment
of erythrocyte membranes with alpha-toxin resulted in the biphasic
formation of 1,2-diacylglycerol and PA as well as an increase of
inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-
4,5-bisphosphate (PIP2) within 30 s.The formation of PA in the first
and secondphases was stimulated by AlF4
- and/or GTP [γS] .
The former was inhibited by phorbol ester and stimulated by protein
kinase C inhibitor; however, these agents had no effect on thesecond
phase . It therefore seems that thetoxin-induced formation of PA
in the first phase is stimulated by endogenous PLC which is activated
by G-protein and is inhibited by protein kinase C, and the formation
of PA inthe second phase is stimulated by endogenous phospholipase
D which is activated by G-protein, but is not controlled by protein
kinaseC . Treatment of rabbit erythrocytes simultaneously with
neomycin resulted in inhibition of the toxin-induced formation of PA
and hemolysis. Furthermore, GTP [γS] stimulated the toxin-induced
formation of PA and hemolysis, and GDP [βS] inhibited them in a
dose-dependent manner . Accordingly, it seems likely that the
toxin-induced formation of PA is tightly linked to hemolysis elicited by
the toxin. Enzymatic activities of alpha-toxin are essential for hemolysis
. We have reported that the PLC activity of the toxin plays a role in the activation of endogenous PIP2-specific PLC in rabbit erythrocytes,
which stimulates the glycerophospholipid system in the membrane,
and that activation of the system leads to hemolysis of the erythrocytes
. The mechanism of the formation of PA is thought to be as shown
in Figure 3. First, hydrolysis of membrane phospholipids by PLC
activity of the toxin causes activation of G-protein. Second, activation
of endogenous PI-PLC and PLD via G-protein activated by the toxin
results in stimulation of PA formation, which leads to hemolysis of
rabbit erythrocytes. These results demonstrate that the toxin-induced
hemolysis is due to activation of phospholipid metabolism systems
through GTP-binding proteins.
||Figure 3: Relationship between phospholipid metabolism and biological
activities of alpha-toxin.
|Activation of sphingomyelin metabolism through GTPbinding
protein induced by alpha-toxin in rabbit erythrocytes
|Alpha-toxin induces hemolysis of rabbit erythrocytes through
the activation of glycerophospholipid metabolism [19-21]. Sheep
erythrocytes contain large amounts of sphingomyelin (SM) but not
PC. Alpha-toxin simultaneously induced hemolysis and a reduction in
the levels of SM in the membrane and an increase in ceramide and
sphingosine 1-phosphate (S1P) . It seemed that the toxin-induced
hemolysis of sheep erythrocytes is closely related to the hydrolysis of
SM in the membrane by the SMase activity of alpha-toxin. Moreover,
the level of phosphorylcholine markedly increased in the cells treated
with the toxin, compared with that of ceramide. PC was not detected
in sheep erythrocytes. These results show that the phosphorylcholine
released is mostly derived from SM. The levels of phosphorylcholine
released by treatment with the toxin were significantly higher than
those of ceramide, suggesting that ceramide is rapidly metabolized
to sphingosine. However, a markedly lower level of sphingosine,
compared with S1P, was detected, although S1P increased with an
increase in the dose of the toxin. It therefore is likely that sphingosine
is rapidly metabolized to S1P in the cells treated with the toxin
. N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the
toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. The data show that the agent specifically
suppresses ceramidase to block hemolysis. Furthermore, DL-threodihydrosphingosine
and B-5354c, isolated from a novel marine
bacterium, both sphingosine kinase inhibitors, blocked the toxininduced
hemolysis and production of S1P and caused sphingosine to
accumulate. S1P potentiated the toxin-induced hemolysis of saponinpermeabilized
erythrocytes but had no effect on that of intact cells
suggesting that intracellular S1P is important for the toxin-induced
hemolysis and that the hemolytic effect is not dependent on the action
of S1P outside the cells. These observations indicate that S1P plays an
intracellular role in the toxin-induced hemolysis, not an extracellular
one, suggesting that it functions as a second messenger in the process.
S1P itself caused no hemolysis of saponin-permeabilized cells,
suggesting that hemolysis may be induced by a combination of S1P and
other events elicited by the toxin in the cells  (Figure 3). GTP[γS]
stimulated alpha-toxin-induced hemolysis, hydrolysis of SM, and
formation of S1P in sheep erythrocytes. It therefore appears that the
activation of GTP-binding proteins is required in the toxin-activated
SM metabolic system. Preincubation of lysated sheep erythrocytes with
pertussis toxin blocked the alpha-toxin-induced formation of ceramide
from SM, suggesting that alpha-toxin activates endogenous SMase
through a pertussis toxin-sensitive Gi type GTP-binding protein. In
addition, incubation of C. botulinum C3 exoenzyme-treated lysates
of sheep erythrocytes with alpha-toxin caused an accumulation of
sphingosine and inhibition of the formation of S1P, indicating that
sphingosine kinase is controlled by Rho (small molecular GTPbinding
protein) in the cells. Therefore, it appears that Rho activated
directly or indirectly by alpha-toxin stimulates sphingosine kinase.
These observations suggest that the alpha-toxin-induced hemolysis of
sheep erythrocytes is dependent on the activation of the SM metabolic
system through GTP-binding proteins, especially the formation of
S1P .The level of C24:1-ceramide was highest among the ceramides
with an unsaturated bond in the fatty acyl chain in the detergentresistant
membranes (DRMs). The toxin specifically bound to DRMs,
resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM)
in DRMs  (Figure 3). Treatment of the cells with pertussis toxin
(PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide
from C24:1-SM in DRMs and hemolysis, indicating that endogenous
sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide is
controlled by a PT-sensitive GTP-binding protein in membranes.
In summary, alpha-toxin hydrolyzes the unsaturated SM, especially
C24:1-SM, by activating endogenous SMase through Gi in the DRMs
of sheep erythrocyte membranes, indicating that alpha-toxin activates
endogenous SMase specific for C24:1-SM. C24:1-ceramides are rapidly
metabolized to sphingosine in the cells treated with the toxin, implying
that the toxin activates ceramidases which selectively recognize
unsaturated ceramides as a substrate . These results show that
the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an
important role in the toxin-induced hemolysis of sheep erythrocytes
|Activation of TrkA signaling through GTP-binding protein
induced by alpha-toxin
|Alpha-toxin stimulated adhesion to the matrix and the generation
– in rabbit neutrophils due to the formation of DAG through
activation ofendogenous PLC by a PT-sensitive GTP-binding protein
[26,27]. Treatment of the cells with the toxin resulted in tyrosine
phosphorylation of TrkA (nerve grown factor high-affinity receptor)
. Anti-TrkA antibody inhibited the productionof O2
– and binding of
the toxin to the TrkA. It has been reported that the phosphatidylinositol
3-kinase (PI3K) signaling pathway has an important role in several effector functions including the generation of O2
− . PI3K is
known to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3),
which is recognized by a pleckstrin homology domain identified as a
specialized lipid-binding module . Several studies have reported
that 3-phosphoinositide-dependent proteinkinase 1 (PDK1) requires
PIP3 as its activator for effective catalytic activity . The toxin
induced phosphorylation of PDK1. K252a, an inhibitor of the TrkA
receptor and LY294002, an inhibitor of PI3K, reduced the toxininduced
production of O2
– and phosphorylation of PDK1, but not the
formation of DAG. The result shows that the toxin-induced activation
of PI3K occurs upstream of the phosphorylation of PDK1, which is
an important step in the toxin-induced generation of O2
− . It is
likely that the toxin-induced phosphorylation of PDK1 is a process
independent of the toxin-induced formation of DAG. Alpha-toxin
induced phosphorylation of PKCθ and PKCζ/λ, and the generation
− induced by the toxin was inhibited by rottlerin and calphostin
C, an inhibitor of PKCθ. We reported that the formation of DAG
induced by alpha-toxin in rabbit neutrophils plays an important role
in the generation of O2
− . K252a and LY294002 inhibited the toxininduced
phosphorylation of protein kinase Cθ (PKCθ). It therefore
appears that the toxin-induced generation of O2
− is dependent on
the activation of PKCθ, through binding of PKCθ phosphorylated
by PDK1 to DAG . U73122, a PLC inhibitor, and pertussis toxin
inhibited the toxin-induced generation of O2
– and formationof DAG,
but not the phosphorylation of PDK1. These observations show that the
toxin independently induces production of DAG throughactivation of
endogenous PLC and phosphorylation of PDK1 via the TrkA receptor
signaling pathway and that these events synergistically activate PKCθ
in stimulating an increase in O2
– . In addition, several studies have
reported that the activation of PKC by various stimuli results in the
generation of O2
− via the activation of mitogen-activated protein kinase
(MAPK) systems [30-32]. The toxin causes phosphorylation of ERK1/2,
but not p38 and SAPK/JNK, implying that the process is dependent
on a MAPK system containing MEK1/2 and MAPK/ERK1/2, but not
systems containing p38 and SAPK/JNK. We showed the participation of
MAPK-associated signaling events via activation of PKCθ in the toxininduced
generation of O2
–. In conclusion, we demonstrated that alphatoxin
induces formation of DAG through the activation of endogenous
PLC by a PT-sensitive GTP-binding protein and phosphorylation
of PDK1 via stimulation of the TrkA receptor, so that DAG and
PDK1 synergistically activate PKCθ, and that the activation of PKCθ
stimulates generation of O2
– through MAPK-associated signaling
events in rabbit neutrophils  (Figure 3). To clarify the mechanism
responsible for the toxin-induced activation of TrkA, the effect of alphatoxin
on TrkA in PC12 cells, a model for studying the differentiation
of neuronal cells in response to TrkA , was investigated. The
toxin induced neurite-outgrowth and phosphorylation of TrkA in
the cells in a dose-dependent manner . TrkA inhibitor K252a and
shRNA for TrkA inhibited the toxin-induced neurite-outgrowth, and
phosphorylation of TrkA and ERK1/2. Furthermore, the binding of
the toxin to PC12 cells transfected with TrkA-specific shRNA vectors
decreased, compared with that to the intact cells, suggesting that the
binding of the toxin to TrkA and the activation of TrkA are required
for neuritogenesis. Nerve growth factor (NGF), which binds to TrkA, is
reported to be required for the differentiation and survival of nerve cells
. Several studies have reported that neurite-outgrowth induced by
NGF is dependent on the activation of MAPK systems [36-38]. Alphatoxin
caused the phosphorylation of ERK1/2, but not that of p38 and
SAPK/JNK, and PD98059, an inhibitor of Erk1/2 cascade, attenuated
the neurite-outgrowth and the phosphorylation of ERK1/2 induced by
alpha-toxin. K252a inhibited the phosphorylation of ERK1/2. These
results showed that the toxin-induced neurite-outgrowth is dependent on activation of the ERK1/2 pathway via TrkA . The wild-type
toxin induced the formation of DAG, and neurite-outgrowth, but
H148G, a variant toxin which binds to cell membranes and has lost the
enzymatic activity, did not. Therefore, it appears that the phospholipid
metabolism caused by the enzymatic activity of alpha-toxin plays an
important role in the activation of TrkA-ERK1/2 signal transduction
and neurite-outgrowth. We demonstrated that the phosphorylation
of TrkA through the phospholipid metabolism induced by the toxin
synergistically plays a key role in neurite-outgrowth  (Figure 3).
|C. perfringens alpha-toxin is an important agent of gas gangrene.
Alpha-toxin induces the hemolysis of rabbit erythrocytes and the
generation of superoxide anion in rabbit neutrophils through the
activation of endogenous PLC via a PT-sensitive GTP-binding protein,
Gi. On the other hand, the toxin also induces the hemolysis of sheep
erythrocytes through the activation of endogenous SMase via Gi and
Rho. The enzymatic activity of the toxin is essential for activation of
phospholipid metabolism via G protein.
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