Reactive Oxygen Species Induced by Shear
Stress Mediate Cell Death in Bacillus subtilis
Susmita Sahoo,1 K. Krishnamurthy Rao,2 G.K. Suraishkumar3
1
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai,
Mumbai, India
2
School of Biosciences and Bioengineering, Indian Institute of Technology Bombay,
Powai, Mumbai, India
3
Biotechnology Department, Indian Institute of Technology Madras, Chennai 600036
India
Abstract: Exposure of Bacillus subtilis to a shear rate of
1,482/s leads to a rapid loss of cell viability after 10 h of
growth. Biochemical and molecular evidences provided
below strongly suggest that cell death under high shear
results from an apoptosis-like process similar to that
described in eukaryotes, with activation of a caspase-3like protease (C3LP) followed by DNA fragmentation.
Shear stress leads to an increase in specific intracellular
reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS
precedes the activation of C3LP and DNA fragmentation,
thus establishing siROS as the molecular link between
shear stress and apoptosis-like cell death. A model is
proposed in which NOX is viewed as being strategically
placed on the plasma membrane of B. subtilis that senses
and converts a mechanical force arising from shear stress
into a chemical signal leading to activation of C3LP, DNA
fragmentation, and thus, apoptosis-like cell death.
Keywords: shear rate; apoptosis-like cell death; C3LP;
DNA fragmentation; siROS; NADH oxidase
INTRODUCTION
Apoptosis or programmed cell death is a genetically
determined process of cellular suicide that is activated in
response to stress or damage, as well as to developmental
signals as observed in multicellular organisms (Rice and
Bayles, 2003). Apoptosis has been primarily described in
eukaryotes, and the two main intracellular indicators of
apoptosis are the activation of the caspase family of proteases
as well as DNA fragmentation that is observed as a ladder in
an agarose gel electrophoresis (Darzynkiewicz and Traganos, 1998; Harvey and Kumar, 1998). In contrast, little is
known about the mechanics of cell death in prokaryotes.
Nevertheless, the possibility of the existence of apoptosislike cell death in bacteria has been suggested in several
bacteria such as E. coli, (Aizenman et al., 1996; Heidrich
et al., 2001), Streptomyces antibioticus (Miguélez et al.,
1999), Staphylococcus aureus (Brunskill and Bayles, 1996),
Bacillus subtilis (Jolliffe et al., 1981; Smith et al., 2000),
Myxococcus xanthus (Rosenbluh and Rosenberg, 1990;
Wireman and Dwokin, 1977), and Xanthomonas campestris
(Gautam and Sharma, 2002a) as well as reviewed by several
authors (Bayles, 2003; Hochman, 1997; Jensen and Gerdes,
1995; Lewis, 2000; Sigler et al., 1999; Yarmolinsky, 1995).
In several of these studies, apoptosis appears to take the
form of cell autolysis that has been suggested to play an
important role in processes such as lysis of the mother/sister
cell during sporulation of B. subtilis (Gonzalez-Pastor et al.,
2003; Smith et al., 2000), and lysis of the fruiting body of M.
xanthus (Rosenbluh and Rosenberg, 1990). In E. coli and S.
aureus, cell lysis after exposure to antibiotics (Heidrich et al.,
2001; Rice et al., 2003; Sat et al., 2001), or other harsh
environmental conditions such as high temperature, DNA
damaging agents, and oxidative stress (Amitai et al., 2004;
Hazan et al., 2004) have been suggested to be caused after
induction of the programmed-cell-death pathways. Hazan
et al. (2004), mention that the death of the cells is activated
only during the logarithmic growth phase, and the death
triggering mechanism remains inactive during the stationary
phase. Nevertheless, there are contrasting views concerning
the nutritional stress-induced cell death observed in stationary phase; Rice and Bayles (2003), reason it as an
example of apoptosis-like cell death, whereas Nystrom
(2003) concluded it to be a stochastic deterioration rather
than programmed cell death. Though occurrence of bacterial
autolysis/apoptosis-like cell death under starvation has been
suspected (Lewis, 2000; Rice and Bayles, 2003), no studies
are available which establish a direct relationship between
apoptosis-like cell death and any stress condition in
prokaryotes.
In the present work, we have studied the relationship
between shear stress, an important form of stress that impacts
cells in a bioreactor, and cell death of B. subtilis. B. subtilis
was chosen as the model system, because it is one of the
preferred hosts in the bio-industry; further, it has been a good
model for the genetic studies of gram-positive bacteria. In the
past few years, the bacterium has been extensively studied in
the context of bio-film formation, cell death, and sporulation
under various stress conditions, but not under shear stress. In
our study, we show that shear stress results in an apoptosislike cell death that is preceded by activation of a caspase-3
like protease as well as DNA fragmentation, events that are
characteristic of eukaryotic apoptosis. We further show that
the apoptotic events occur as a result of an increase in reactive
oxygen species (ROS) induced by shear stress; ROS
regulation of prokaryotic apoptosis-like cell death has not
been reported earlier.
MATERIALS AND METHODS
Organism and Growth Conditions
B. subtilis 168 (trpC2) (Bacillus Genetic Stock Center) cells
were grown in Luria-Bertani (LB, pH 7.0) medium at 378C.
The pre-inoculum was prepared by growing cells in liquid
media in a shake flask for 12 h at 190 rpm. These were diluted
1:10 (v/v) in to fresh media and transferred to either a couette
flow bioreactor (CFB, working volume 250 mL) or a shake
flask (working volume 100 mL) for applying the desired
shear stress. The initial viable cell concentration was
maintained approximately at 4.5 106 0.9 106 cfu/mL
by adjusting the pre-inoculum concentration. Viable cell
concentrations expressed as colony forming units per
milliliter (cfu/mL) were determined by plating on standard
LB agar plates. These experiments were repeated five times,
and each point shown represents the mean value.
The CFB is a device that provides defined shear rates, and
thus the effect of shear rate on cells can be studied
conveniently using the CFB. In a typical stirred bioreactor
the shear environment is highly undefined, and the cells
would experience a wide range of shear stresses. Thus it
cannot be used to study the effect of shear on cells. The design
and operating conditions of CFB is discussed in detail in a
related work (Sahoo et al., 2003). In brief, different shear
rates, ranging from 222/s to 1,482/s, were achieved in the
CFB with the help of two concentric cylinders, of which the
inner one is stationary and the outer one rotates. The shear
rate of 1,482/s was the highest shear rate that we could
reliably obtain in the CFB, and hence it was chosen for
studying the molecular aspects. The inner cylinder is perforated and covered with teflon (polytetrafluoroethylene, PTFE)
membrane to provide oxygen to the culture in the annular
space The volumetric oxygen transfer rates (kLa values) in
the CFB and shake flask (500 mL capacity, 190 rpm) were
comparable. Thus, differential oxygen availability cannot be
the reason for the difference in cell responses discussed
below. In addition, the average shear rate obtained in the
shake flask was calculated to be 0.028/s, which is negligible
compared to the shear rates obtained in the CFB. Therefore,
shake flask was established to be an appropriate control for
shear studies in CFB (Sahoo et al., 2003).
Samples were collected from both the CFB and the shake
flask cultures at regular intervals. Cells were separated from
the supernatant by centrifugation (10,000g for 10 min, unless
specified otherwise), and stored in a deep freezer (208C) until
further analysis was done. For measuring intracellular ROS
levels, and for NADH oxidase (NOX) assay, the samples were
processed immediately. The above experiments were repeated
at least twice for reproducibility. The maximum variation was
about 13%, and the errors are indicated in the figures.
Enzymatic Assays
The NADH oxidase (NOX) activity in B. subtilis was
measured spectrophotometrically as the reduction of NADH
at 340 nm (Morre, 2002). Treatment with diphenyleneiodonium chloride (DPI), an inhibitor of NOX, was accomplished
by exposing cells to 0.6 mM DPI (Sigma Chemical Co., St.
Louis, MO) for 5 min at 9 h of cultivation, cells separated by
centrifugation, and re-suspended in LB of similar nature
without DPI. Lactate dehydrogenase activity was measured
spectrophotometrically by the oxidation of NADH in the
presence of pyruvate as previously described (Yoshida and
Freese, 1975). The enzyme units were calculated from a
standard graph prepared from LDH (Hi-media, Mumbai,
India). Caspase-3-like protease (C3LP) activity from B.
subtilis cell lysates was measured using a synthetic
fluorogenic substrate, Ac-DEVD-AMC (Sigma Chemical
Co.), as previously described (Gautam and Sharma, 2002a).
The enzyme units were calculated from a standard graph
prepared using human caspase-3 (Sigma Chemical Co.). The
enzyme units were obtained per 108 live cells [U/(108 live
cells)] by normalizing the total enzyme units in 1 mL with the
corresponding viable cell concentrations.
Treatment Procedure for Caspase-3 Inhibitors
Two different types of human caspase inhibitors were used
in the experiments, one broad-based pan-caspase inhibitor,
Z-VAD-fmk (Sigma Chemical Co.) (50 mM), and specific
caspase-3 inhibitor, Ac-DEVD-CHO (Sigma Chemical Co.)
(50 mM) were used. The shear-stressed cells at 8 h of
cultivation (before the increase of caspase and ROS-time
profile determined previously in separate experiments) were
exposed to the inhibitors for 5 min. The cells were separated
by centrifugation and re-suspended in LB of similar nature
without the treatment agent. Before the Ac-DEVD-CHOtreatment, the cells were permeabilized with permeabilization buffer (Kohler et al., 2002; McKenna and Cotter, 2000).
The toxic levels of the inhibitors were calculated from the
viability analysis of shake flask cultures treated with different
concentrations of the inhibitors.
Anti-Caspase-3 Application
To determine the neutralizing activity of human anti-caspase3 (from rabbit, IgG fraction, Sigma Chemical Co.), 1 mg of
the protein was incubated with different concentrations of
human caspase-3 enzyme (Sigma Chemical Co.) for 1 h at
258C. Human caspase-3 activity was assayed in the above
mixture as described before, and compared with the
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corresponding control samples without the anti-caspase-3.
Similarly, to measure the neutralizing activity of the B.
subtilis C3LP, cell extracts from sheared cultivation was
incubated with human anti-caspase-3 in the reaction buffer
(of caspase-3 assay), and C3LP activity was compared with
the corresponding control. The isotype control (rabbit IgG,
Sigma Chemical Co.) was treated with the cell extracts at the
same concentration as the anti-caspase-3. The concentration
used for anti-caspase-3 was determined through titration.
Determination of Specific Intracellular ROS (siROS)
Intracellular ROS was measured and identified to be
superoxide radical by ESR spectroscopy using the spin trap
Dimethyl-1-pyrroline-N-oxide (DMPO, Sigma Chemical
Co.) as previously described (Sahoo et al., 2004). A known
standard curve of superoxide radical (using potassium
superoxide, KO2, Sigma Chemical Co.) was used (Valentine
et al., 1984) to determine the concentrations. The specific
intracellular ROS level was obtained by normalizing the
intracellular ROS concentrations with the corresponding
viable cell concentrations. The ROS type was identified by a
protocol previously described (Sahoo et al., 2004). Induction
of ROS was achieved by treatment of cells with 2.5 mM H2O2
at pulsed intervals of 30 min for the entire cultivation period
(Sahoo et al., 2004). The cultivation was continued in dark to
prevent H2O2 degradation.
DNA Extraction, Agarose Gel
Electrophoresis, and Western Blot
Standard methods for DNA extraction and agarose gel
electrophoresis were used (Sonenshein, 2000). A 10 mL
sample of the cell culture was used to extract DNA, the purity
and concentration of which were checked using a spectrophotometer. Equal amounts of DNA from each sample was
electrophoresed in 1% agarose gel in TBE buffer at 80 mA for
1–2 h. The gels were stained with ethidium bromide and
photographed under UV light.
For Western blot analysis, the cells were lysed, and 40 mg
of each protein sample (determined by Bradford’s method)
were separated in 15% SDS–polyacrylamide gel (PAGE).
The proteins were transferred to nitrocellulose membrane
(Hybond-P, Amersham-Pharmacia, Piscataway, NJ) for incubation in primary antibody at 48C, followed by extensive
washing in PBS and incubation with peroxide-conjugated
secondary antibody. The antigen was detected using NBTBCIP chromogenic substrate (Roche, Nutley, NJ). Appropriate controls were run for each experiment, which are
discussed in the results and discussion section.
RESULTS AND DISCUSSION
Shear Stress Induces Cell Death
To study the effect of shear stress on the growth of B. subtilis,
the concentration of viable cells in a couette flow bioreactor
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(CFB) was determined as a function of time, under different
shear rates. The growth rates obtained at different shear rates
were: 0.22/h at 0.028/s, 0.26/h at 445/s, 0.33/h at 741/s, and
0.76/h at 1,482/s (Sahoo et al., 2003). Figure 1 presents time
profiles of viable cell concentrations when cultivated at a
maximum shear rate of 1,482/s, and at a negligible shear rate
of 0.028/s (when grown in a shake flask). In subsequent
sections, the latter is considered as the control, and the same
has been established as a suitable control in our earlier work
(Sahoo et al., 2003). At a shear rate of 1,482/s, the viable cell
concentration increased to a maximum at 10 h, and thereafter
rapidly decreased by 86% within the next 2 h (at 12 h of
cultivation), and by greater than 99% in 15–18 h of
cultivation. Since the decrease in viable cell concentration
from the maximum value (in the cultivations under shear)
is by several orders of magnitude, the representation of
decrease in viable cell concentration in percentages does not
effectively communicate the reduction. For example, the
decrease in viable cell concentration from the maximum
value was calculated to be 86% at 12 h and 99.94% at 15 h.
The value 86% does not appear to be significantly different
from 99.94%, even though the difference between the two
viable cell concentrations is three orders of magnitude.
Therefore, to quantify the decrease better, we have defined
RVC, the ratio of viable-cell concentrations, that is
RVC ¼
Viable cell concentration at a given time
Maximum viable cell concentration
In terms of RVC, the decrease in viable cell concentration at
the shear rate of 1,482/s at 12, 15, and 18 h was calculated to
be 1.5 101, 6 104, and 3.1 106, respectively. On the
contrary, control cells achieved the maximum viable cell
concentration between 10–12 h of growth, and that remained
almost constant until at least 18 h (RVC ¼ 9 101). The
significant rapid decline in cell viability (RVC) was also
observed at shear rates ranging from 222 to 1,111/s (Fig. 2).
Figure 1. Growth profiles of B. subtilis under two different shear rates:
1,482/s (bold circles) and 0.028/s (open circles). Viable cell concentrations
expressed as colony forming units per milliliter (cfu/mL) were determined on
standard LB agar plates. The experiments were repeated five times, each
point shown represents the mean value with the error bars (not visible due to
the logarithmic scale).
Figure 2. Effect of shear rates on cell viability (expressed as RVC*). The
viable cell number (colony forming units, cfu) was determined by the
standard plate count on LB agar at 378C. Each number is the average value of
three different experiments. The maximum variation was 8%. The error bars
are not visible due to the logarithmic scale. * RVC ¼ Viable cell concentration at a given time/maximum viable cell concentration.
The data provided here indicates that the loss of cell viability
could be directly related to the applied shear stress.
We were thus interested in determining the mode of cell
death under shear stress. A possible reason for cell death is
that mechanical forces arising from shear stress could lead to
necrosis and cell rupture. Necrosis is a form of degenerative
cell death resulting in a progressive cell rupture and release of
intracellular contents (Darzynkiewicz and Traganos, 1998).
To determine if cell death was due to necrosis, we measured
the release of an intracellular enzyme, lactate dehydrogenase
(LDH, a marker for necrosis (Essmann et al., 2003)), in to
the medium from cells cultivated at a shear rate of 1,482/s.
If cell death was due to necrosis resulting in progressive
cell rupture, one would expect to see a progressive and
cumulative increase in LDH levels in the medium as death
sets in. The results in Figure 3 clearly show that, although cell
viability is significantly reduced after 10 h, there is no
significant release of the enzyme until about 16 h, and the
enzyme levels are comparable to the control. Thereafter,
between 16 and 18 h, there is an increase in LDH levels,
probably indicating that the cells that had died earlier,
eventually ruptured. We thus believe that the type of cell
death that occurs under shear stress is unlikely to be due to
necrotic cell lysis.
Further, as the shear stressed cells attained a cell
concentration almost 100-fold higher than the control cells
(in the same medium, LB), it can be argued that the difference
in the residual content of the spent-media or, in other words,
starvation, induced after the maximum growth was achieved,
induced the cell death. This possibility was examined by two
different tests. In the first test, the cells were grown under
shear stress (1,482/s) till the maximum viable cell concentration was reached (10 h). Then, they were harvested from
the suspected-spent-media, transferred to fresh LB media,
cultivated under both high shear stress (1,482/s) and negligible shear stress (0.028/s) conditions, and the cell viability
measured. Interestingly, in both the cases, the cells continue
Figure 3. LDH and C3LP enzyme activity in B. subtilis at shear rates of
1,482/s and 0.028/s. LDH was measured in the extracellular medium to
measure cell rupture. C3LP was measured endogenously. Both LDH and
C3LP at different time points in shear stressed cells (1,482/s) are joined with
solid lines.
to show a rapid reduction in viability (RVC, 15 h ¼ 3 104
and 2 104 respectively). These results suggest that the
program for cell death was already established during the
growth phase, and the cells were thus committed to die. In
the second test, the cells after 10 h of growth in the shake flask
were harvested, suspended in the spent-media of the shearstressed cells (cell-free media extracted after the cells were
grown in the reactor for 10 h, at 1,482/s), and cultivated under
both high shear stress and negligible shear stress conditions.
Remarkably, the cells did not undergo rapid cell death
in either case (RVC, 15 h ¼ 0.6 and 0.9, respectively)
immediately. Nevertheless, under shear stress, the cells
started dying after 8 h growth in the spent-media, which was
comparable to the cell death after 10 h in fresh LB media
under stress. In all these experiments, the initial cell concentration was maintained approximately at the maximum cell
density attained by shear-stressed cells (5 1011 cfu/mL).
These experiments suggest that the cell death under shear
involves a certain types of ‘irreversible’ program inside the
cell, induced during the active-growth-phase, to bring out its
own demise, regardless of the external conditions following
the growth phase. Therefore, starvation as a principal cause
for cell death was ruled out.
In addition, an adaptive response also does not seem to be
responsible for the observations. We have shown in a related
earlier study (Sahoo et al., 2004), that the transcription factor
(sB) and the general stress protein (Ctc), which regulate/
induce adaptive response in B. subtilis under other types of
stress, are maintained at significantly low levels (40-fold
lower) under high shear stress compared to controls. More
importantly, the well known adaptive mechanism, sporulation, which is known to be activated under different stresses,
remained inactive under shear stress. This may possibly
imply an inactive adaptive response mechanism under shear
stress.
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We therefore, examined the possibility that shear stress
leads to apoptosis-like cell death in B. subtilis.
Shear-Induced Cell Death
Resembles Eukaryotic Apoptosis
In eukaryotes, apoptosis is characterized by two major
events: activation of a family of proteases known as caspase,
which is considered as the main executer of apoptosis
(Harvey and Kumar, 1998), and DNA fragmentation
(Darzynkiewicz and Traganos, 1998). We thus decided to
investigate if similar events occurred in B. subtilis upon
encountering shear stress.
Caspase-3-Like Protease (C3LP) is Activated by
Shear Stress and is Responsible for Cell Death
In eukaryotes, at least 14 members constitute the caspase
family of proteases. Five of these, caspases 3, 6, 7, 8, and 9,
are actively involved in the caspase cascade of different
forms of apoptosis (Sauerwald et al., 2003). Of these
caspases, the presence and involvement of caspase-3-like
activity has been previously demonstrated in Xanthomonas
campestris (Gautam and Sharma, 2002a). We were thus
interested in determining if B. subtilis contained an
endogenous C3LP activity that is activated under shear
stress. C3LP enzyme activity was measured in shear stressed
(1,482/s), and control cells with a fluorogenic substrate, AcDEVD-AMC [N-acetyl-asp-glu-val-asp-AMC (7-amino-4methylcoumarin)] that is specific to caspase-3 (Gautam and
Sharma, 2002a; Nicholson and Thronberry, 1997; Nicholson
et al., 1995). Figure 3 shows that in shear-stressed cells, the
C3LP activity rapidly increased at 9 h of growth, reached a
maximum at 10 h [1.1 U/(108 live cells)], and declined to a
basal level at 12 h. In contrast, control cells exhibited a basal
level of activity [0.2 U/(108 live cells)] throughout growth.
Thus, the result suggests that B. subtilis contains a caspase-3
like protease that is dramatically increased under shear stress.
Maximal activation of C3LP is achieved at 10 h, and it
precedes the death phase (Fig. 3) suggesting that C3LP
activation is necessary for cell death. Indeed, when an
irreversible inhibitor of caspase, Z-VAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl (b-methyl ester)-fluoro-methyl
ketone) (Kohler et al., 2002; Sauerwald et al., 2003), was
applied to cells under shear stress at 8 h and C3LP activity
measured at 10 h, the activity was reduced to levels similar to
that of control cells (Fig. 4). This reduction in C3LP was
accompanied by a dramatic increase in the concentration of
viable cells at 15 h, as indicated by a RVC (15 h) increase
from 6 104 in absence of the inhibitor to 3 101 in
presence of inhibitor (Fig. 4). Similar experiments with
another inhibitor of caspase-3 group of enzymes, Ac-DEVDCHO, showed consistent results—the RVC at 15 h increased
from 6 104 in the absence of the inhibitor to 3 102 with
Ac-DEVD-CHO (Fig. 4). In addition, no C3LP enzyme
activity was detected, when the healthy cells were grown in
the spent-media from reactor, confirming our contention that
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Figure 4. Effect of caspase inhibitors on maximum C3LP activity and 15 h
viable cell concentration: maximum C3LP activity (black bars), and 15 h
viable cell concentrations (white bars) at 1,482/s in absence of inhibitors, and
in presence of Z-VAD-fmk and Ac-DEVD-CHO.
shear-induced-starvation could not be a reason for apoptosislike-cell death in B. subtilis under shear stress.
Our suggestion that B. subtilis possess a caspase-3 like
protease is based on the use of a specific substrate. To more
directly show that this indeed is the case, we measured C3LP
activity in the presence of an antibody to human caspase-3
that was added to cell lysates of B. subtilis grown until 10 h
under shear stress (1,482/s). The capability of anti-human
caspase-3 IgG to neutralize caspase-3 activity was initially
determined on human caspase-3 and found to inhibit its
activity by 74 10% (data not shown). Figure 5 shows that
addition of rabbit anti-human caspase-3 IgG to a B. subtilis
lysate resulted in an 85 4% reduction in C3LPs activity as
compared to less than 5% reduction in activity with a nonspecific IgG. These results suggests that B. subtilis does
contain a caspase-3-like protease.
Figure 5. Effect of human anti-caspase antibody on maximum C3LP
activity of B. subtilis: Enzyme activity measurements in cell lysates from
cultures grown at shear rate of 1,482/s for 9 h treated with anti-caspase-3 IgG
(black bar), with non-specific IgG (gray bar) and without IgG (white bar).
The presence of C3LP protein was confirmed from the
Western blot analysis using the human caspase-3 antibody
(Fig. 6). As is evident from the blot, a strong hybridization
signal was obtained from the shear stressed cells (1,482/s,
lane 1, 2, and 3), indicating the presence of an endogenous
caspase-3-like protein. The protein was not detected in the
control cells (lane 4). An experimental control with a nonspecific IgG did not produce any hybridization signal. Thus,
the presence of a caspase-3-like protein in B. subtilis under
shear stress is confirmed. The molecular weight of the protein
was found to be around 55 kDa (Fig. 6), which appeared in the
cells after about 5 h of growth under shear stress, maximized
at 9 h (as observed from the intensity), and disappeared after
10 h (was not detected in the subsequent samples). The
maximum enzyme activity of the protein, however, correlated with the onset of cell death, at 9 h. This time lag between
the appearance of the protein and its enzyme activity suggests
the presence of a precursor protein or an inactive proenzyme,
which probably, undergoes processing to form an active
caspase-3-like enzyme. This notion was proved by the
application of z-VAD-fmk, which is known to inhibit
the activation or the processing of human caspase-3 (Slee
et al., 1996). When the shear-stressed cells were cultivated
with the caspase inhibitor, Z-VAD-fmk, the level of the
protein at 9 h remains almost same, as shown by the intensity,
where as, the enzyme activity decreased. Our results strongly
suggest that B. subtilis does contain a caspase-3 like protease,
which is activated under shear stress, and probably initiates
events that lead to cell death; nevertheless, we do not rule out
the possibility that other caspases-like proteases may also be
involved in the same cell death process. The B. subtilis C3LP
was found to be identical in size to the caspase of another
unicellular prokaryote, X. campestris, each approximately
55 kDa, in contrast to 32 k Da of human caspase. This provides an important clue to the evolution of PCD among the
unicellular forms, specifically caspases, which are possibly
structurally and functionally conserved across the organisms.
Further, to identify the bacterial C3LP gene, we carried
out a sequence homology search with the human caspase-3
c-DNA, and the caspase-3 protein; the caspase-3 sequence
has not yet been identified in Xanthomonas or in other
bacterial species. But, the study did not yield any significant
sequence similarity for the construction of a possible
B. subtilis C3LP-mutant.
Shear Stress Induces Fragmentation of DNA
Another key marker of apoptosis is the fragmentation of
chromosomal DNA that is visualized as a ladder of bands on
an agarose gel electrophoresis (Darzynkiewicz and Traganos, 1998). To further support the contention that shear stress
leads to apoptosis-like cell death, we determined whether
shear-stressed cells lead to fragmentation of DNA. The
results in Figure 7 show that DNA fragmentation was not
observed in shear stressed cells at 9 h (lane 1) but was clearly
visible at 13 h (lane 2) and clear DNA laddering represented
by a smear followed by high molecular weight fragments of
DNA (more than 100 kbp) was seen at 15 h (lane 3). Although
DNA fragmentation is usually seen as a smear in eukaryotic
apoptosis, clear fragmentation as observed in this study has
been reported in certain cell lines such as thymocytes,
lymphocytes, and mouse cell lines (Brown et al., 1993;
Lagarkova et al., 1995), where fragments of 50–700 kbp
were observed. On the contrary, the control cells did not show
any DNA fragmentation even at 15 h (lane 4). Interestingly,
the inhibition of C3LP activity with Z-VAD-fmk in cells
subjected to shear stress, completely abolished DNA
fragmentation (lane 7) indicating that activation of C3LP is
necessary for DNA fragmentation.
Protein Synthesis is Required for Cell Death
Apoptosis is a genetically regulated program that is
dependent on active protein synthesis (M.O. Hengartner,
2000), and as suggested previously, requires the synthesis of
autolytic enzymes (Lewis, 2000). Thus it would be expected
that an inhibitor of protein synthesis would rescue the cells
from death, by inhibiting the synthesis of autolytic enzymes.
Figure 6. Western blot analysis for caspase-3-like protease (C3LP) under different shear rates. The caspase-3-like protein was detected in the cells grown
under shear stress 1,482/s at different time points (lane 1, 2, and 3), whereas in control cells (0.028/s), the protein was not detected (lane 4). Lane 5 shows the
reduction in caspase-3-like protein when the caspase inhibitor z-VAD-fmk was applied under shear stress (1,482/s). Each lane was loaded with 40 mg protein.
The size of B. subtilis C3LP under shear stress is found to be approximately 50–55 KD (positions of markers indicated at the right). The corresponding C3LP
enzyme activities (as enzyme units) are indicated below each lane.
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Figure 7. Effect of shear on DNA fragmentation in B. subtilis at shear rates
of 0.028/s and 1,482/s with and without treatment of cells with H2O2, DPI,
and Z-VAD-fmk.
Figure 8 presents the effect of a well-known protein synthesis
inhibitor in bacteria, chloramphenicol (2 mg/mL), on the
viable cell concentration of B. subtilis in shear stressed
(1,482/s) cells. Application of chloramphenicol at 9 h of
cultivation decreased the drop in viable cell concentration
from its maximum value at 17 h; the viable cell concentration
decreased from a maximum value of 8.5 1011 cfu/mL, to
about 3 1011 cfu/mL with chloramphenicol (2 mg/mL),
compared to about 5 108 cfu/mL in the absence of the
antibiotic. This corresponds to an increase in the RVC:
3.5 101 at 17 h in the presence of 2 mg/mL chloramphenicol compared to 6 104 in the absence of the antibiotic.
This observation that functional protein synthesis is required
for the apoptosis-like cell death. Interestingly, cell death
could not be inhibited when the inhibitor was applied at 10 h
Figure 8. Viable cell concentrations (cfu/mL) of B. subtilis during
cultivations at shear rate of 1,482/s (bold circles), with chloramphenicol
(2 mg/mL) treatment at 9 h (open circles) and with chloramphenicol (2 mg/
mL) treatment at 10 h (open triangles). Each point is representative of three
different experiments; the mean values are presented. Error bars (maximum
12%) are not visible due to the logarithmic scale.
124
of cultivation (Fig. 8) supporting an earlier conclusion that at
10 h the cells are already committed to die.
In addition to the above biochemical and molecular
characteristics, several morphological alterations were also
observed in shear-stressed cells similar to those seen in
eukaryotic apoptosis. These changes included reduction in
cell size (Sahoo et al., 2003), and unequal cell division (data
not shown). Similar reduction in cell size has been reported in
several eukaryotic apoptosis (Darzynkiewicz and Traganos,
1998) and prokaryotic apoptosis-like cell death (Gautam and
Sharma, 2002b).
Thus, the biochemical, molecular, and morphological
evidences taken together strongly argue in favor of an
apoptosis-like cell death in B. subtilis in response to shear
stress. This process requires a functional protein synthesizing
machinery. Cell death appears to be preceded by activation of
a caspase-3-like protease followed by DNA fragmentation,
the key events previously seen in eukaryotes. Moreover,
apoptosis-like cell death was associated with suppression of
the sporulation process under high shear stress (Sahoo et al.,
2004).
Shear Stress Activates NADH Oxidase in
B. subtilis Resulting in Increased Reactive
Oxygen Species
Having established that shear stress leads to apoptosis-like
cell death, we were interested in investigating the link that
connects these two events. We had previously reported that
shear stress results in the production of intracellular reactive
oxygen species (ROS). They were identified as a superoxidederived radical (Sahoo et al., 2003), and the precursor
superoxide radicals are most likely generated by the activity
of a plasma membrane bound enzyme, NADH-oxidase
(NOX) (Morre, 2002). NOX is known to occur in eukaryotes,
although its presence in B. subtilis had not been previously
established. We were thus interested in determining if a
NOX-like activity existed in B. subtilis. The results in
Figure 9 show that NOX was present at a basal level of 0.11
U/(108 live cells) in control cells that was considerably
increased to 0.58/(108 live cells) at a shear stress of 1,482/s.
This increase was concomitantly accompanied by a 11-fold
increase in specific intracellular ROS level (siROS) from
0.4 mmol/(108 live cells) to 4.3 mmol/(108 live cells). The
increase in siROS was maximal at 9 h of growth as shown
earlier (Sahoo et al., 2003). Furthermore, treatment of cells at
shear stress of 1,482/s with diphenyleneiodonium chloride
(DPI), a specific inhibitor of NOX, resulted in reduction of
NOX activity as well as the levels of siROS to that seen in
control cells (Fig. 9). Corresponding increases in NOX and
siROS levels were also observed at other shear stress levels
(data not shown). Our results thus suggest the existence of a
NOX-like enzyme whose activity is dramatically increased
under shear stress, which results in the formation of siROS.
NOX activity was assayed using the whole cells, and
therefore, its presence is suspected in the plasma membrane
of B. subtilis. Existence of NOX-like enzymes in B. subtilis
Intracellular ROS Activates
Caspase-3-Like Proteases
Figure 9. Measurement of NOX activity (black bars), siROS level (white
bars) and viable cell concentration (gray bars) in absence and presence of
DPI and H2O2. The viable cell concentration varies inversely to the NOX
activity and siROS concentration. The values of siROS are indicated above
the corresponding white bars. DPI (0.6 mM) was exposed to the sheared-cells
at 9 h for 5 min, and 30 min pulses of 2.5 mM H2O2 was applied to the cells
under negligible shear stress.
was not reported earlier. Furthermore, as siROS levels reach a
maximum at 9 h and cell death occurs after 10 h of growth, it
suggests that siROS is the link between shear stress and
apoptosis. To further establish the link between siROS and
apoptosis-like cell death, we determined the effect of
quenchers and inducers of ROS on cell death, C3LP activity,
and DNA fragmentation.
Increased siROS Leads to Apoptosis-Like
Cell Death
Cell Death is Reversed With DPI in Shear-Stressed
Cells and Augmented in H2O2-Treated Control Cells
To address the question whether the formation of ROS under
high shear (1,482/s) is responsible for cell death, DPI, the
inhibitor of NOX activity, was added to cells grown under
high shear at 8 h, and cell viability was measured at 15 h. The
results in Figure 9 show that DPI application enhanced the
RVC to 6 101 from 6 104 in the absence of DPI at 15 h.
Thus, reduction of siROS in shear-stressed cells to that of
control levels was able to prevent cell death, supporting the
notion that the heightened level of siROS under shear stress is
the cause of cell death. In further support of this contention,
treatment of control (shake flask) cells with H2O2, a very well
known inducer of ROS, that increased siROS from 0.4 to 1.1
mmol/(108 live cells) resulted in cell death (RVC ¼ 5 103)
as compared to almost no cell death (RVC ¼ 9 101) in the
absence of H2O2 (Fig. 9).
Thus the results clearly confirm that the increased levels of
siROS generated in shear-stressed cells leads to apoptosislike cell death. It is not clear at present whether this is a direct
effect or the initial steps in a cascade that eventually
culminate in cell death.
To further delineate the involvement of siROS in mediating
apoptosis-like cell death, we measured intracellular C3LP
activity in shear stressed cells (1,482/s) treated with DPI at
8 h. Figure 10, clearly shows that application of DPI leads to a
fourfold decrease in C3LP activity, reaching an activity
similar to that seen in control cells. In contrast, cells grown in
shake flask under negligible shear (0.028/s), and treated with
H2O2 showed a threefold increase in C3LP (Fig. 10). These
results thus show a clear correlation between increased
siROS levels and C3LP activity. As siROS levels peaked at 9 h
and C3LP activity is observed maximally at 10 h, it is very
likely that siROS is responsible for the activation of C3LP. In
support of this view, measurement of siROS in shear-stressed
cells in the presence of a broad based inhibitor of caspase,
Z-VAD-fmk, resulted in an inhibition of C3LP without any
effect on the siROS levels (data not shown).
Specific Intracellular ROS Induces
DNA Fragmentation
To determine whether increased siROS levels are also
responsible for DNA fragmentation, shear stressed cells
were treated with the NOX inhibitor, DPI, at 8 h, and DNA
fragmentation was assessed at 15 h. Figure 7 clearly shows
that in the presence of DPI, DNA fragmentation is almost
completely abolished (lane 6) as compared to that seen in its
absence (lane 3). On the other hand, cells grown under
negligible shear in which DNA is intact (lane 4), when treated
with H2O2 at 5 h, show considerably enhanced DNA
fragmentation at 15 h (lane 5). Interestingly, inhibition of
C3LP activity with the caspase inhibitor Z-VAD-fmk, results
in a dramatic reduction in DNA fragmentation (lane 7)
suggesting that DNA fragmentation is an event that is
downstream to the activation of C3LP. These results once
again clearly show that the siROS that is induced under shear
Figure 10. Maximum caspase-3-like protease activity at 1,482/s (white
bar), at 1,482/s with DPI (dark gray bar), at 0.028/s (black bar), and at 0.028/s
with H2O2 (light gray bar).
125
stress leads to apoptosis-like cell death through activation of
C3LP followed by DNA fragmentation.
An examination of the timing of ROS production, C3LP
activation, and DNA fragmentation in shear-stressed cells
reveals that siROS induction is a relatively early event (about
9 h of cultivation) that is followed by rapid activation of
C3LP, which peaks at 10 h and declines by 12 h. This is then
followed by initiation of DNA fragmentation at 13 h, and an
overt DNA laddering at 15 h (Fig. 7). Thus, the induction of
superoxide-derived radical and DNA fragmentation are
temporally separated by a window of 4–6 h. This suggests
that the induced siROS is unlikely to directly act on the
chromosome leading to the fragmentation of DNA; instead,
siROS lies upstream in a pathway that first involves C3LP
activation followed by DNA fragmentation. This is supported
by the observation that inhibition of C3LP activity leads to
significant reduction in DNA fragmentation (Fig. 7, lane 7).
Our results thus suggest that shear induced siROS function as
a primary messenger of apoptosis-like cell death in B.
subtilis, rather than a secondary effector as has been observed
in certain kinds of apoptosis (Higuchi, 2003).
Interestingly, data from the control cultures until 36 h show
that although minor loss of viability (0.6% at 17 h, and 4% at
30 h; insignificant in terms of RVC values) was observed, it
was not associated with the characteristic increase in
caspase-3 like protease, or intracellular ROS, in contrast to
the cells exposed to shear stress.
On the basis of the results presented in the article, we
present a hypothesis for shear stress-induced apoptosis-like
cell death in B. subtilis (Fig. 11). We propose that shear stress
activates NADH oxidase (NOX), probably located at the
plasma membrane, resulting in the formation of an
intracellular superoxide-derived radical. This, in turn,
activates a caspase-3-like protease (C3LP) that leads to,
eventually, DNA fragmentation and cell death. As NOX is a
membrane bound protein (Baker et al., 1998), its activation
suggests a mechanism whereby mechanical stress resulting
from shear is converted into biochemical signals.
Figure 11. Temporal sequence of events, and a model for the mechanism
of shear-induced apoptosis-like cell death in B. subtilis.
126
CONCLUSIONS
Shear stress has been shown to cause apoptotis-like cell death
in a prokaryote, B. subtilis. Shear stress caused increase in
specific intracellular ROS levels, which preceded caspase-3
like protein activation and DNA fragmentation, the molecular hallmarks of eukaryotic apoptosis. A model for
apoptosis-like cell death due to shear stress in B. subtilis
has also been proposed.
We acknowledge Tushar Beuria for providing the non-specific IgG for
our study, and the Sophisticated Analytical Instruments Center, IIT
Bombay, for the use of the ESR spectrometer.
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