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Molecular and Cellular Basis of Microvascular Perfusion
Deficits Induced by
Clostridium perfringens
and
Clostridium septicum
Michael J. Hickey
1
*, Rain Y. Q. Kwan
1
, Milena M. Awad
2
, Catherine L. Kennedy
2
, Lauren F. Young
3
, Pam
Hall
1
, Leanne M. Cordner
2
, Dena Lyras
2
, John J. Emmins
3
, Jul ian I. Rood
2
1 Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia, 2 Australian Bacterial Pathogenesis
Program, Department of Microbiology, Monash University, Victoria, Australia, 3 Department of Immunology, Monash University, Alfred Medic al Research and Education
Precinct, Prahran, Victoria, Australia
Abstract
Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by
Clostridium perfringens. The C. perfringens a-toxin has been shown capable of inducing these changes, but its potential
synergy with perfringolysin O (h-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative
agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this
study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional
microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant
microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis
of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell
death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary
perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion,
consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the a-toxin or
perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic
complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise
correlating with the level of the C. septicum a-toxin. Together, these data indicate that as a result of its ability to produce a-
toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular
perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is
central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium.
Citation: Hickey MJ, Kwan RYQ, Awad MM, Kennedy CL, Young LF, et al. (2008) Molecular and Cellular Basis of Microvascular Perfusion Deficits Induced by
Clostridium perfringens and Clostridium septicum. PLoS Pathog 4(4): e1000045. doi:10.1371/journal.ppat.1000045
Editor: Michael S. Gilmore, Schepens Eye Research Institute, United States of America
Received December 20, 2007; Accepted March 14, 2008; Published April 11, 2008
Copyright: ß 2008 Hickey 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.
Funding: This work was supported by funding from the Australian National Health and Medical Research Council (NHMRC) awarded to MJH (Program Grant
#334067) and JIR (Program Grant #284214). MJH is an NHMRC Senior Research Fellow.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: michael.hickey@med.monash.edu.au
Introduction
Gas gangrene is a life-threatening syndrome most commonly
associated with invasion of tissue by the anaerobic bacterium,
Clostridium perfringens type A. The pathology of g as gangrene is hi ghly
complex, but is tho ught to be mediated by disruptions in tissue
perfusion, associated with alterations in platelet aggregation and
leukocyte margination [1,2]. Histological assessment of infected
tissues, both in humans and experimental animals, has revealed a
characteristic pattern of extensive myonecrosis, edema, thrombosis,
and restriction of leukocyte infiltration to the perivascular regions in
the infected site [1,3]. Toxins produced by C. perfringens type A have
been shown to be essential to the development of this pathology [2-7].
a-toxin and perfringolysin O are the major toxins produced by type A
strains but they are known to produce other extracellular toxins and
enzymes including a collagenase, the cysteine protease a-clostripain,
sialidases and hyaluronidases [8,9]. Toxin production in C. perfringens
is regulated in response to environmental or growth phase signals by
the VirSR two-component signal tran sduction system [9].
Preparations containing the C. perfringens a-toxin, which has
phospholipase C and sphingomyelinase activity [10], and
perfringolysin O, a cholesterol-dependent cytolysin, also known
as h-toxin [11], have been shown to induce rapid and irreversible
reductions in muscle blood flow, via generation of intravascular
platelet-leukocyte aggregates [6]. In vivo and in vitro studies
implicate platelet adhesion glycoproteins GPIIb/IIIa and P-
selectin (CD62P) as contributing to these heterotypic aggregates.
However, the precise mechanism of action of these toxins,
particularly in combination, requires further investigation.
Examination of the vascular effects of purified clostridial toxins
has generated findings that emphasize the complexity of the tissue
response. Purified a-toxin has been shown to promote myonecrosis
and disrupted perfusion within minutes of application to a muscle,
responses which correlate with the histopathological appearance of
affected tissue [7,12]. Furthermore, mutation of the a-toxin gene,
plc, is associated with more effective leukocyte recruitment to the
site of C. perfringens infection, suggesting a critical role for this toxin
PLoS Pathogens | www.plospathogens.org 1 April 2008 | Volume 4 | Issue 4 | e1000045
in limiting leukocyte recruitment [3,4]. In contrast, a-toxin has
been shown to promote adhesion of leukocytes to cultured
endothelial cells and to induce leukocyte recruitment and
expression of endothelial adhesion molecules in vivo [13]. Similarly,
mutation of the perfringolysin O structural gene, pfoA, has been
shown to allow increased leukocytic infiltration into infected tissues
[3]. These findings suggest that the a-toxin alone is insufficient to
account for the inability of leukocytes to efficiently access C.
perfringens-infected tissue and raise the possibility that the complete
complement of C. perfringens toxins is necessary for the most
damaging response. This concept is supported by several studies
that indicate that a-toxin and perfringolysin O act synergistically,
such that the most severe pathology is only observed if both toxins
are present [3,5,14]. If so, then experiments with purified toxins
may not accurately reflect the complex pathology associated with
clostridial infection.
Virulence studies in the mouse myonecrosis model using mutant
C. perfringens strains lacking a-toxin and perfringolysin O have
provided clear evidence of the roles of these toxins in promoting
myonecrosis [3–5]. However, the effects of these mutant strains on
myonecrosis are yet to be correlated with effects on the muscle
microcirculation, as assessed using real-time in vivo imaging.
Moreover, another clostridial species, Clostridium septicum, can also
cause myonecrotic tissue infections, including atraumatic or
spontaneous gas gangrene [15,16]. C. septicum also produces an
a-toxin, which does not have phospholipase C activity, and is not
related to the a-toxin of C. perfringens; it is a pore-forming cytolysin
encoded by the csa gene. The virulence of C. septicum is chiefly
attributable to its a-toxin [17]. However, the pathological effects of
C. septicum on the microcirculation, and the potential function of
the a-toxin in this area, have not been examined. Therefore, the
aim of this study was to investigate the cellular and molecular basis
of vascular disruption associated with exposure to products of
clostridial strains capable of inducing myonecrotic infections. This
objective was achieved using intravital microscopy to quantitate
microvascular perfusion and cellular injury during exposure to
supernatants from cultures of C. perfringens and C. septicum. Use of
mutant clostridial strains deficient in various toxins enabled the
relative roles of these gene products to be assessed. This study
represents the first time this integrated approach has been used in
the analysis of the pathological response to toxins released by C.
perfringens and C. septicum.
Results
Mechanisms of C. perfringens-mediated reduction in
microvascular perfusion
In initial experiments, we quantitated the effect of culture
supernatants from a wild-type, virulent C. perfringens type A strain,
JIR325, on microvascular perfusion in the cremaster muscle. Prior
to commencing supernatant superfusion, functional capillary
density averaged approximately 75 mm/mm
2
(Figure 1). After
60 min superfusion with the Trypticase-peptone glucose (TPG)
culture medium (diluted 1:1 in bicarbonate superfusion buffer),
microvascular perfusion was not significantly altered from basal
levels (p = 0.2). In contrast, superfusion with similarly diluted
culture supernatant from strain JIR325 caused a marked reduction
in microvascular perfusion. After 60 min, functional capillary
density was significantly reduced (p,0.001) to 15-40% of the level
at the start of the experiment (Figure 1A). Additional experiments
were performed to examine the time course of the reduction in
capillary perfusion in more detail. After 30 min exposure to C.
perfringens supernatant, mean functional capillary density was not
significantly reduced from baseline levels (Figure 1B), although in
some animals at this time, blood flow in cremasteric arterioles was
inconsistent and pulsatile, and regions of arteriolar constriction
were apparent (data not shown). In contrast, by 60 min, perfusion
was significantly reduced relative to both 0 and 30 mins.
Figure 1C shows representative images of the microcirculation
after 60 min superfusion with either TPG or JIR325 supernatant,
demonstrating perfused microvessels labeled with sodium fluores-
cein, and the severe reduction in microvascular perfusion after
60 min exposure to supernatant from JIR325. It was notable that
in supernatant-exposed tissue, fluorescein remained visible in
larger arteries, but was rarely observed in capillaries, indicating
that smallest elements of the vasculature were most affected by the
defect in perfusion at this time point. Videos showing microvas-
cular perfusion in representative experiments from mice exposed
to TPG (Video S1) or JIR325 (Video S2) are available on-line in
the Supporting Information section.
To assess the possibility that the reduction in microvascular
perfusion induced by C. perfringens supernatant was associated with
cell death, propidium iodide (PI) staining was used to identify
irrevocably injured cells in the cremaster muscle. Exposure of the
tissue to absolute ethanol at the end of the experiment
demonstrated that this staining approach was capable of
identifying dead cells (Figure 2A, B). Superfusion with TPG
medium induced minimal cell death ( Figure 2). However, in
muscles superfused with JIR325 supernatant, the number of PI-
stained cells was significantly increased within 30 min, with no
further increase observed at 60 min (Figure 2A). Notably, the
number of PI-stained cells observed after treatment with JIR325
was greater than 50% of those ultimately stained following
exposure of the tissue to ethanol (Figure 2A, B). Therefore, it was
concluded that treatment with C. perfringens culture supernatant led
to substantial cell death.
Previous studies have implicated alterations in neutrophil
margination and platelet aggregation in C. perfringens-mediated
blood flow alterations [7,18]. To assess these factors in this model
system, mice were depleted of either neutrophils or platelets, using
Author Summary
Clostridial myonecrosis is a life-threatening process in-
duced by infection with species such as C. perfringens and
C. septicum. The associated pathology includes muscle
death and a characteristic disruption in tissue perfusion.
Exotoxins produced by these species have been implicated
in the reduction in perfusion. However, how these toxins
function in tandem remains unclear. In this study we used
intravital microscopy to study microvascular blood flow in
a muscle exposed to products of C. perfringens and C.
septicum. C. perfringens supernatants induced cellular
injury and a progressive reduction in blood flow. Removal
of blood-borne platelets and neutrophils from the
circulation reduced the alteration in blood flow. In
addition, this response was reduced by genetic deletion
of either the a-toxin or perfringolysin O, providing the first
indication that each of these exotoxins contributes to the
reduction in blood supply to affected tissues. Using a
similar approach, we observed that C. septicum superna-
tant induced a comparable reduction in perfusion, which
was mediated in part via the C. septicum a-toxin. These
results indicate that platelets, neutrophils and multiple
clostridial toxins contribute to reduced blood supply and
oxygen delivery associated with clostridial infection and
suggest that the dominant component of the pathology is
toxin-induced cellular injury and death.
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 2 April 2008 | Volume 4 | Issue 4 | e1000045
anti-Gr-1 or anti-platelet serum respectively, prior to undergoing
exposure to culture supernatant. These approaches have been
validated previously in this laboratory as achieving greater than
95% depletion of their respective targets over a similar time course
to that used in the current experiment [19]. In mice depleted of
Figure 1. Effect of
C. perfringens
supernatant on microvascular
perfusion in the mouse cremaster muscle. A: Functional capillary
density was assessed in cremaster muscles under basal conditions, and
after 60 min of superfusion with either TPG medium (as control, n = 6)
or filtered supernatant from C. perfringens (JIR325, n = 6). Each data
point indicates an individual animal. Horizontal bar denotes group
mean. * denotes p,0.001 versus basal data using paired t-test. NS
denotes not significant. B: Time course of reduction in perfusion in
muscles exposed to C. perfringens supernatant. Microvascular perfusion
was quantitated prior to, and after 30 and 60 min exposure to JIR325
supernatant. Data are displayed as the 30 & 60 min functional capillary
density readings expressed as a percentage of the baseline reading for
each individual animal. ** denotes p,0.01 versus baseline data, and t
denotes p,0.02 versus 30 min (using paired t-test). C: Representative
images displaying microvascular perfusion, as demonstrated by
presence of infused sodium fluorescein within microvessels. Images
are shown for mice exposed to either TPG or JIR325 supernatants, both
prior to and after 60 min of supernatant exposure.
doi:10.1371/journal.ppat.1000045.g001
Figure 2. Effect of
C. perfringens
supernatant on cell viability in
the mouse cremaster muscle. A: Cell death was assessed via
propidium iodide (PI) staining in cremaster muscles superfused with
either TPG medium (as control, n = 3) or filtered supernatant from C.
perfringens (JIR325, n = 3). Images were captured at 0, 15, 30 and 60 min
of superfusion. To assess the validity of this approach and determine
the maximal degree of PI staining, at the end of the experiment muscles
were treated with absolute ethanol and PI staining re-assessed. B:
Representative images of PI-stained muscles superfused with either TPG
or JIR325, taken (i) after 60 min supernatant superfusion, and (ii) after
subsequent ethanol superfusion. * denotes p,0.05 versus TPG-treated
animals.
doi:10.1371/journal.ppat.1000045.g002
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 3 April 2008 | Volume 4 | Issue 4 | e1000045
either neutrophils or platelets, the reduction in microvascular
perfusion induced by JIR325 supernatant was significantly attenu-
ated (Figure 3). These data directly implicate these blood-borne
cells in contributing to C. perfringens-induced microvascular collapse.
To determine which C. perfringens toxin(s) were responsible for
the reduction in microvascular perfusion, supernatants were
prepared from isogenic C. perfringens strains genetically deficient
in the production of either a-toxin (plc mutant, JIR4107) or
perfringolysin O ( pfoA mutant, JIR4069) [4] or both toxins
(JIR4444) [5]. The toxin levels for each of these strains are shown
in Table 1, confirming the absence of activity of the respective
toxins. However, it is notable that the a-toxin activity in the
perfringolysin O-mutant JIR4069 was lower than that in the wild-
type strain (Table 1). Animals treated with a-toxin-deficient
supernatant (JIR4107) displayed a significant attenuation in the
normal C. perfringens-induced perfusion deficit (Figure 4). Simi-
larly, supernatants from the perfringolysin O mutant (JIR4069)
resulted in an almost complete abolition of the microvascular
collapse seen in response to superfusion with supernatants from
wild-type C. perfringens (Figure 4). The absence of both a-toxin
and perfringolysin O (JIR4444) resulted in a minimal reduction in
microvascular perfusion (Figure 4)&(Video S3, Supporting
Information section), comparable to that observed following
superfusion with medium alone (Figure 1). These data suggest
that both a-toxin and perfringolysin O are capable of inducing
microvascular perfusion collapse within 60 minutes.
The roles of the individual C. perfringens toxins were investigated
in more detail using supernatants from complemented C. perfringens
strains. These strains were generated from the double-mutant
JIR4444 such that either perfringolysin O (JIR4460) or a-toxin
(JIR4461) were now expressed [5]. Assays of toxin activity in these
strains confirmed complementation of the respective toxin to levels
comparable to those in the wild-type strain (Table 1). In this
assay, complementation with the a-toxin resulted in a significant
reduction in perfusion relative to that seen with supernatants from
the double mutant (JIR4444) (Figure 4 ). Indeed, the reduction in
perfusion induced by JIR4461-derived, a-toxin
+
supernatants was
not statistically different from that induced by supernatants from
wild-type C. perfringens (JIR325). In contrast, complementation with
perfringolysin O alone resulted in a variable response which was
not significantly altered from that seen in response to the double
mutant (Figure 4).
C. septicum supernatants also cause reduction in
microvascular perfusion
C. septicum is a primary contributor to the aetiology of
atraumatic gas gangrene [15]. However, its effects on microvas-
Figure 3. Roles of neutrophils and platelets in
C. perfringens
-
induced perfusion defect. Groups of mice were either left untreated
(n = 6), or depleted of either neutrophils (anti-Gr1, n = 5) or platelets
(anti-platelet serum, n = 8) prior to exposure of the cremaster muscle to
C. perfringens (JIR325) supernatant. Subsequently, functional capillary
density was quantitated 60 min after commencing superfusion. Data
are also shown for mice following 60 min superfusion of TPG alone (first
column). t denotes p,0.05 versus TPG. * denotes p,0.01 versus JIR325
alone.
doi:10.1371/journal.ppat.1000045.g003
Figure 4. Comparison of perfusion defect induced by supernatants from genetically altered
C. perfringens
strains.Supernatantswere
generated from the following C. perfringens strains: wild-type JIR325 (a
+
h
+
); a-toxin-deficient JIR4107 (a
2
h
+
); perfringolysin O-deficient JIR4069 (a
+
h
2
);
double toxin-deficient JIR4444 (a
2
h
2
); perfringolysin O-complemented JIR4460/a
2
h
2
(ph
+
) (where p denotes gene inserted via a shuttle plasmid); and a-
toxin-complemented JIR4461/a
2
h
2
(pa
+
) (n = 6/group). Supernatants were applied to the cremaster muscle as previously described. Functional capillary
density measurements were made prior to, and after 60 min of supernatant superfusion. Data are displayed as the 60 min functional capillary density
readings expressed as a percentage of the baseline reading for each individual animal. Data are also shown for TPG alone (first column). * denotes p,0.001
versus JIR325. t denotes p,0.05 versus TPG. ** denotes p,0.05 versus JIR4444. y denotes NS between JIR325 and JIR4461.
doi:10.1371/journal.ppat.1000045.g004
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 4 April 2008 | Volume 4 | Issue 4 | e1000045
cular perfusion have not been examined. Therefore, we used a
comparable approach to that used with C. perfringens, and applied
supernatants of wild-type C. septicum (JIR6086) to the cremasteric
microvasculature. Similar to the response induced by C. perfringens,
culture supernatants from C. septicum induced a significant
microvascular perfusion deficit over a 60 min time course
(Figure 5). To assess the role of the C. septicum a-toxin (a different
protein than the similarly named toxin secreted by C. perfringens),
we compared the perfusion deficit induced by wild-type superna-
tant with that induced by supernatant from a csa mutant (JIR6111)
deficient in a-toxin production, and with an isogenic a-toxin-
complemented derivative (JIR6146). The relative a-toxin titres for
these three strains are shown in Table 2. No detectable hemolytic
activity was observed for the a-toxin mutant (JIR6111) on horse
blood agar. However, as previously observed [17], a-toxin activity
was restored to levels above wild-type in the strain complemented
for a-toxin (JIR6146), presumably reflecting the multi-copy nature
of the complementation plasmid. Exposure of tissues to superna-
tants from the a-toxin mutant resulted in a mean perfusion at
60 min of 29.467.3 mm/mm
2
, versus 17.366.5 mm/mm
2
in
response to wild-type C. septicum supernatant (p = 0.057, when
analyzed as % reduction from baseline) (Figure 6). In contrast, in
tissues exposed to supernatants from the a-toxin-complemented
strain (JIR6146), microvascular perfusion was almost entirely
absent by 60 min, a substantially greater effect than that seen in
response to the wild-type C. septicum supernatants (Figure 6). This
differential response may be explained by the higher a-toxin titre
in the complemented strain (Table 2).
Discussion
The pathology of clostridial myonecrosis is complex and unique.
Existing work has demonstrated a role for the cessation of
microvascular blood flow in addition to the poorly understood
phenomenon of inhibition of leukocyte entry into infected tissues,
termed leukostasis [3,6]. The aim of this study was to use high
resolution intravital microscopy to examine the microcirculation
directly during exposure to culture supernatants from wild-type
and toxin-mutant clostridial strains, and to quantitatively assess
microvascular perfusion. The findings indicate that toxins of both
C. perfringens and C. septicum induce rapid and severe reductions in
blood flow, observations that correlate with the findings of our
previously published examination of the ability of these bacteria to
rapidly induce myonecrosis [3–5,17]. Visualization of the
microcirculation indicated that blood flow in capillaries was
severely reduced. Given the central role of these vessels in the
delivery of oxygen and nutrients, this effect would be expected to
be an important contributor to the necrosis that occurs in infected
tissue. Cell death, as assessed by PI incorporation, was found to
occur prior to substantial reduction in tissue perfusion, raising the
Table 1. Relative toxin activities in supernatants from wild-type and mutant C. perfringens cultures
Strain Genotype Ref Toxin status
a
a-toxin (units mg
21
protein)
(x 10
3
) Perfringolysin O (log
2
(titre))
JIR325
b
Wild-type [34] Plc
+
PFO
+
22.763.9
c
6.560.3
JIR4107 JIR325plc::ermB [4] Plc
2
PFO
+
,0.9
d
5.760.1
JIR4069 JIR325pfoA::ermB [4] Plc
+
PFO
2
9.164.5 ,1.0
d
JIR4444 JIR4069 plcVpJIR1774 [5] Plc
2
PFO
2
,0.9 ,1.0
JIR4460 JIR4444(pJIR871) [5] Plc
2
PFO
+
,0.9 6.560.1
JIR4461 JIR4444(pJIR1642) [5] Plc
+
PFO
2
18.162.7 ,1.0
a
Plc signifies a-toxin, PFO signifies perfringolysin O
b
JIR325 is a rifampicin and nalidixic acid resistant derivative of strain 13, a human gangrene isolate.
c
Data are shown as mean6SEM of at least two independent cultures
d
Limit of detectability
doi:10.1371/journal.ppat.1000045.t001
Figure 5. Effect of
C. septicum
supernatant on microvascular
perfusion in the mouse cremaster muscle. Functional capillary
density was assessed in cremaster muscles prior to, and after 60 min of
superfusion with filtered supernatant from C. septicum (JIR6086, n = 7).
* denotes p,0.001 versus basal data using paired t-test.
doi:10.1371/journal.ppat.1000045.g005
Table 2. Relative C. septicum a-toxin (Csa) activity in
supernatants from wild-type and mutant C. septicum cultures
Strain Genotype Ref
Toxin
status
a
C. septicum
a-toxin
(log
2
(titre))
JIR6086
b
Wild-type [17] a-toxin
+
4.0
JIR6111 JIR6086csaVermB [17] a-toxin
2
,1
c
JIR6146 JIR6111(pJIR2503) [17] a-toxin
+
7.0
a
C. septicum a-toxin
b
JIR6086 is a rifampicin resistant derivative of strain BX96; the clinical status of
which is not known
c
Limit of detectability
doi:10.1371/journal.ppat.1000045.t002
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 5 April 2008 | Volume 4 | Issue 4 | e1000045
possibility that this injury was an important contributor to the
reduction in perfusion. Platelets and neutrophils were also found to
play a role in the response, as were both the a-toxin and
perfringolysin O of C. perfringens, and the C. septicum a-toxin. These
findings support our earlier demonstration of the functions of these
toxins in inducing clostridial myonecrosis, and provide new
insights into the pathogenesis of this syndrome [3–5,17].
The pathogenic mechanisms of the highly virulent C. septicum
are poorly characterized. Previous studies have demonstrated an
essential role for the C. septicum a-toxin in myonecrosis [17].
However, the effects of this toxin on the microcirculation have not
been examined. Our results provide the first evidence that C.
septicum toxins have the capability of inducing vascular collapse in a
similar manner as toxins from C. perfringens. Furthermore, they
implicate the C. septicum a-toxin as capable of mediating this
process, despite the fact that its structure, specificity and biological
activity are very different from those of any of the toxins produced
by C. perfringens type A [20]. However, it was notable that
supernatants from a-toxin-deficient C. septicum retained the
capability of reducing perfusion by approximately 60%, suggesting
that other C. septicum products such as other hemolysins [21,22],
sialidase [23] or DNAse [24] may have contributed to this
response. Additional experiments are needed to determine if there
are other C. septicum toxin(s) that are involved in mediating the
cessation of microvascular blood flow.
In the response to C. perfringens infection or exposure to C.
perfringens a-toxin, the formation of platelet/leukocyte aggregates
and leukostasis have been implicated as being of central
importance [3,7,12,25]. In addition, C. perfringens a-toxin has been
reported to induce interactions between platelets and leukocytes, a
process that may underlie leukostasis [26]. In the present study,
obvious aggregate formation or thrombosis was not observed in
response to superfusion with C. perfringens supernatants, although
this finding may reflect the different approaches used in these
studies. In the model used by Bryant et al., purified clostridial
toxins caused reductions in blood flow within less than 5 minutes
[7]. Similarly, Alape-Giron et al. observed very rapid (within 1–
2 min) alterations in the morphology of muscle fibers upon
exposure to a-toxin, thrombus formation within 2–5 min, and
complete cessation of blood flow within 15–20 mins [12]. In
contrast, the response in the present study was much more gradual
and associated with minimal changes in muscle morphology. It is
likely that these divergent observations are due to differences in the
toxin activity present in culture supernatants versus preparations
of purified a-toxin. Despite this difference, platelet depletion was
partially protective against the reduction in blood flow induced by
C. perfringens supernatants. These data are consistent with previous
findings in which the anti-thrombotic agent heparin was protective
against a-toxin-induced vascular collapse [7], and support the
hypothesis that platelets are important contributors to the C.
perfringens-induced deficit in microvascular perfusion.
The role of leukocytes is one of the least well understood aspects
of this response. The inability of leukocytes to effectively infiltrate
Clostridium-infected tissues is a hallmark of the disease pathology
[3], but it remains unclear if this is a contributing factor to
myonecrosis or an epiphenomenon. Nonetheless, examination of
the microvasculature has shown that neutrophil depletion protects
against reductions in blood flow induced by C. perfringens a-toxin
[7]. This finding is supported by the results of the present study, in
which neutrophil depletion was protective against the microcircu-
latory collapse induced by C. perfringens supernatants. In studies of
ischemia/reperfusion injury, the neutrophil has been shown to
contribute to reductions in microvascular perfusion via luminal
obstruction of microvessels [27–29]. In addition, a-toxin, via
platelet GPIIb/IIIa and P-selectin (CD62P), can induce formation
of platelet-leukocyte aggregates, which could also act to reduce
microvascular perfusion [7,26]. These processes provide potential
mechanisms for the contribution of neutrophils to the defect in
microvascular perfusion induced by C. perfringens. However, these
data must also be viewed in light of recent work examining the
effect of neutrophil depletion on C. perfringens-induced myonecrosis.
O’Brien et al. showed that in animals infected with large inoculates
of C. perfringens, neutrophil depletion failed to protect against
myonecrosis [18]. This finding indicates that under conditions
where large numbers of live bacteria are present, C. perfringens is
capable of inducing fatal myonecrosis irrespective of the potential
perfusion-maintaining effect afforded by neutrophil depletion.
Many studies implicate a-toxin as being of central importance
in C. perfringens–associated myonecrosis [2,4,12,30]. In the present
study, analysis of the molecular basis of the vascular response to C.
perfringens confirmed the previously observed capability of the a-
toxin to induce vascular collapse [7,12]. This was shown using a-
toxin-deficient C. perfringens mutants, which were less effective at
reducing perfusion than wild-type C. perfringens, and by comple-
mentation of an a-toxin and perfringolysin O-deficient strain with
a-toxin, which restored the ability to disrupt perfusion to levels not
different from that of the wild-type strain. However, these
experiments also provided evidence suggesting a role for
perfringolysin O in reducing perfusion. Firstly, supernatants from
perfringolysin O-deficient strains caused substantially less vascular
compromise than those from wild-type strains, although this result
may have been complicated by the comcomitant reduction in a-
toxin activity in this mutant. Secondly, supernatants from a-toxin/
perfringolysin O-double mutant strains complemented with
perfringolysin O were capable of reducing microvascular perfusion
in some mice, a response not seen in response to supernatants
lacking both toxins. These data suggest a novel activity for this
toxin, although further work is required to confirm this hypothesis.
It has been demonstrated that perfringolysin O contributes to the
Figure 6. Role of the
C. septicum
a-toxin in reducing muscle
microvascular perfusion. Supernatants were prepared from wild-
type C. septicum (JIR6086/WT), or genetically-modified C. septicum
strains either lacking the C. septicum a-toxin (JIR6111/csa, n = 7), or
complemented with the a-toxin (JIR6146/csa(pcsa
+
), n = 3). Superna-
tants were applied to the muscle as previously described. Functional
capillary density measurements were made prior to, and after 60 min of
supernatant superfusion. Data are displayed as the 60 min functional
capillary density readings expressed as a percentage of the baseline
reading for each individual animal. Data are also shown for TPG alone
(first column). t denotes p,0.05 versus TPG. * denotes p,0.05 for the
comparisons shown.
doi:10.1371/journal.ppat.1000045.g006
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 6 April 2008 | Volume 4 | Issue 4 | e1000045
escape of C. perfringens from the macrophage phagosome, is a
primary mediator of C. perfringens-mediated macrophage cytotox-
icity, and can activate TLR4, indicating that perfringolysin O can
make important contributions to C. perfringens-induced pathology
independently of the a-toxin [14,31]. However, analysis of the
virulence of mutant C. perfringens strains lacking both a-toxin and
perfringolysin O provides clear evidence that the pathology of C.
perfringens infection is most severe in the presence of both toxins
[3,5,14].
In cases of clinical C. perfringens infection, the toxin levels present
in the tissues are unknown. Given this situation, the relative merits
of examining the effects of purified toxins versus culture
supernatants are difficult to assess. In the present experiments,
the rationale for the use of culture supernatants is that it allowed
assessment of the tissue response to the complete set of clostridial
toxins, an approach that is likely to more accurately model clinical
infection. Furthermore, it also allowed the assessment of the effect
of removal of single C. perfringens gene products from the
supernatant. However, it must be noted that the genetic
approaches used in this study produced some unexpected effects.
In the C. perfringens experiments, the a-toxin activity in the
perfringolysin O mutant JIR4069 was lower than that in the wild-
type strain. Therefore, the possibility cannot be excluded that
alterations in perfusion associated with exposure to this strain are
due not only to the absence of perfringolysin O, but also to its
lower a-toxin activity. Similarly, in the C. septicum studies, plasmid-
mediated complementation of the C. septicum a-toxin mutant
resulted in a greater level of toxin activity than that in the wild-
type strain. Nevertheless, the level of perfusion defect observed in
response to the three C. septicum strains examined correlated with
the level of a-toxin activity, supporting the concept that the C.
septicum a-toxin is capable of inducing a reduction in microvascular
perfusion.
Vascular leukostasis and the absence of neutrophils is a
characteristic of clinical cases of clostridial myonecrosis, but the
mechanisms of leukostasis and its contribution to the process of
myonecrosis remain unclear. Since the initial trauma associated
with C. perfringens gangrene cannot be replicated in experimental C.
perfringens infections, high inoculates are required to induce
myonecrosis; at lower infection rates, neutrophil infiltration
proceeds unhindered [4,18]. Furthermore, purified a-toxin has
been shown capable of inducing adhesion molecule expression and
leukocyte recruitment in a vascular bed in which blood flow was
presumably maintained [13]. From these findings it could be
argued that if neutrophils are able to enter the infected tissue
efficiently before cessation of microvascular perfusion, they are
then able to participate in clearing the infection. However, in the
present study we observed extensive cell death prior to the most
severe reductions in blood flow. It is possible that endothelial cells
were irreversibly injured by exposure to the clostridial toxin
mixtures. This hypothesis is highly relevant to the process of
neutrophil recruitment in that leukocyte transmigration across the
endothelium has been shown to require active responses from
endothelial cells [32,33]. It is likely that severely injured
endothelial cells would be unable to undergo the cytoskeletal
rearrangements necessary to allow transmigration. Therefore, the
possibility that leukostasis in part reflects a reduction in efficiency
of leukocyte transmigration due to endothelial injury cannot be
discounted.
Taken together, these data are consistent with a model of C.
perfringens-induced myonecrosis in which both a-toxin and
perfringolysin O contribute to the reduction in microvascular
perfusion. The data also indicate a potential role for injury of local
cells, in addition to roles for circulating platelets and neutrophils.
Although it was not possible to clarify the influence of C. perfringens
products on leukostasis, it is clear if a comparably severe reduction
in perfusion occurred during clinical infection, then this would
prevent delivery of large numbers of leukocytes to the affected site.
Methods
Antibodies and Reagents
Antibodies used in these experiments were RB6-8C5 (purified
from supernatant) and rabbit anti-thrombocyte serum (Accurate
Chemical & Scientific). Sodium fluorescein, PI and all other
reagents were purchased from Sigma Chemical Co. (St. Louis,
MO), unless otherwise stated.
Bacterial culture and toxin assays
The genotypes and toxin production levels of clostridial strains
are shown in Tables 1 and 2. C. perfringens culture supernatants
were prepared as previously described except that the supernatants
were filter-sterilized [4]. C. perfringens a-toxin and perfringolysin O
were assayed as previously described [34]. C. septicum cultures were
grown in TPG medium to a turbidity of 1.5 at 600 nm, then
centrifuged at 10,0166g for 15 min and the supernatants
collected, filter sterilized, aliquoted and stored at 270uC.
Supernatant samples were then assayed for a-toxin activity as
previously described [17].
Preparation of cremaster muscle for intravital microscopy
Male BALB/c mice were bred in-house (Monash University
Animal Services) and housed in conventional conditions. All
animal experimentation was approved by the Monash University
Animal Ethics ‘B’ Committee. The mouse cremaster muscle was
prepared for intravital microscopy as described previously [35].
Briefly, mice were anesthetized with ketamine hydrochloride
(150 mg/kg; Pfizer, West Ryde, Australia) and xylazine (10 mg/
kg; Troy Laboratories, Smithfield, Australia) by intraperitoneal
injection. The left jugular vein was cannulated to administer
additional anesthetic and fluorochromes. Catheters were not
heparinized to avoid obscuring potential thrombotic effects of
clostridial products. Animals were maintained at 37uCona
thermocontrolled heating pad. The cremaster muscle was
exteriorized onto an optically-clear viewing pedestal. The muscle
was cauterized longitudinally and held flat against the optical
window via attachment of silk sutures. The tissue was kept warm
and moist by superfusion of warmed bicarbonate buffered saline
(pH 7.4), and covered with a coverslip held in place with vacuum
grease.
The cremasteric microcirculation was visualized using an
intravital microscope (Axioplan 2 Imaging; Carl Zeiss, Australia)
with a X 20 objective lens (LD Achroplan 20X/0.40 NA, Carl
Zeiss, Australia) and an X 10 eyepiece. Brightfield images of the
preparation were visualized using a colour video camera (Sony
SSC-DC50AP, Carl Zeiss, Victoria, Australia), and fluorescence
images were visualized using a low-light video camera (Dage-MTI
IR-1000; SciTech, Preston South, Victoria, Australia). All images
were recorded for playback analysis using a videocassette recorder
(Panasonic NV-HS950, Klapp Electronics, Victoria, Australia).
The number of adherent leukocytes within 100 mm post-capillary
venule regions was quantitated as described previously [35]. This
intravital microscopy approach was selected to also enable
examination of the effects of clostridial toxins on leukocyte-
endothelial cell interactions. However, it was found that treatment
of muscle with diluted TPG alone elevated leukocyte interactions
above that in buffer-treated mice. In addition, leukocyte adhesion
in muscles exposed to JIR325 supernatant was not significantly
Clostridium and Microvascular Perfusion
PLoS Pathogens | www.plospathogens.org 7 April 2008 | Volume 4 | Issue 4 | e1000045
increased above that in mice treated with TPG alone (data not
shown). Therefore leukocyte rolling and adhesion were not
assessed in further groups.
Analysis of functional capillary density (FCD)
After a 20 min stabilization period following cremaster
exteriorization, an initial assessment of the microcirculation was
performed in the exteriorized cremaster muscle. Intravenously
administered sodium fluorescein, imaged via epifluorescence
(excitation wavelength–460 nm, emission wavelength–515 nm)
was used to delineate perfused microvessels. A single bolus of
sodium fluorescein (5 mL of 10 mg/ml in saline) was injected
intravenously, and fluorescence images of five adjacent areas of
tissue rapidly recorded on video for subsequent analysis. This low
molecular weight fluorescent marker was selected as it was rapidly
cleared from the circulation thus allowing assessment of the
functional state of the microcirculation at multiple time points in
the same animal. The cremaster was then superfused for 60 min
with supernatant from various strains of C. perfringens or C. septicum,
diluted 1:1 in bicarbonate superfusion buffer, and perfusion re-
assessed at the end of the experiment in identical fashion. In some
experiments, additional readings were performed after 30 min of
supernatant superfusion.
To quantitate functional capillary density, single video frames
were captured using Adobe Premiere software, and functional
capillary density determined using Adobe Photoshop, as previously
described [36]. Briefly, the captured video frames were opened,
converted to RGB mode, and a new layer generated. The Pencil
tool was then used to trace a line (5 pixel width) over the capillaries
containing fluorescent material. The Magic Wand tool was used to
select the traced lines, and the Histogram command used to
quantitate the number of pixels in the traced line. Based on the
width of the traced line and the resolution and magnification of the
image, the length of the perfused capillaries was calculated and
expressed per unit area (mm/mm
2
) [36].
To assess the role of neutrophils in the microvascular perfusion
response, mice were pre-treated with anti-Gr-1 antibody (RB6-8C5,
150 mg/mouse i.p.) 4 hrs prior to the experiment [19]. Similarly, to
examine the role of platelets, platelet depletion was achieved via
injection of rabbit anti-mouse thrombocyte serum (15 mL, i.p.) 4 hrs
before exposure to C. perfringens supernatants [19].
In vivo analysis of cellular injury
Cell death in the exteriorized muscle preparation was assessed
using PI staining, as previously described [37]. PI labels the nuclei
of cells with disrupted cell membranes, but not healthy cells. The
muscle was superfused with PI (1.0 mM, in superfusion buffer) and
PI-stained nuclei visualized via epifluorescence (excitation wave-
length–535 nm, emission wavelength–617 nm). Images of two
randomly-selected regions within the muscle were captured at
defined intervals throughout the experimental period. The
number of PI-positive nuclei per frame was quantified and
expressed as (cells/field).
Statistical Analysis
All data are displayed as mean6SEM. Student’s t tests or one
way ANOVA was performed to compare experimental groups. P
values ,0.05 were considered significant.
Supporting Information
Video S1 Video showing normal microvascular perfusion in the
cremaster muscle 60 mins after commencement of TPG
superfusion. Several areas of muscle are shown shortly after
intravenous administration of sodium fluorescein, which labels
flowing blood vessels. Functional, perfused capillaries can be
detected via the presence of fluorescent (white) material moving
through multiple small caliber vessels. The degree of microvascu-
lar perfusion in this tissue is consistent with complete perfusion of
all capillaries in the muscle.
Found at: doi:10.1371/journal.ppat.1000045.s001 (7.00 MB
MOV)
Video S2 Video showing effect of superfusion with supernatants
from wild-type C. perfringens (JIR325). Several areas of muscle are
shown shortly after intravenous administration of sodium
fluorescein. Sixty min after commencing superfusion, the absence
of microvascular perfusion is manifest as large areas of muscle
containing very few fluorescein-filled microvessels. Sodium
fluorescein-associated fluorescence is only apparent in occasional
larger arterioles and venules, whereas only few capillaries are
perfused. The reduction in the number of perfused capillaries in
this experiment is readily apparent by comparison with that in
muscles superfused with TPG (Video S1).
Found at: doi:10.1371/journal.ppat.1000045.s002 (6.47 MB
MOV)
Video S3 Video showing effect of superfusion with supernatants
from an a-toxin/perfringolysin O mutant (JIR4444) of C.
perfringens. Several areas of muscle are shown shortly after
intravenous administration of sodium fluorescein. After 60 min
of superfusion, substantial capillary perfusion is apparent in these
tissues (demonstrated by the presence of intravenous fluorescein).
Indeed, the degree of capillary perfusion in muscles exposed to
supernatant deficient in a-toxin and perfringolysin O is compa-
rable to that in tissues exposed to TPG (Video S1), indicating the
combined actions of these toxins in promoting loss of perfusion.
Found at: doi:10.1371/journal.ppat.1000045.s003 (5.16 MB
MOV)
Acknowledgments
The authors thank Prof. Richard Boyd for helpful discussions.
Author Contributions
Conceived and designed the experiments: MH MA DL JE JR. Performed
the experiments: RK MA CK LY PH LC. Analyzed the data: MH RK
PH. Wrote the paper: MH MA JR. Supervised experiments: DL JE.
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