Bacillus anthracis spores influence ATP synthase activity in murine macrophages.

Abstract

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be downregulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

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J. Microbiol. Biotechnol. (2008), 18(4), 778–783
Bacillus anthracis Spores Influence ATP Synthase Activity in Murine
Macrophages
Seo, Gwi-Moon1, Kyoung Hwa Jung1, Seong-Joo Kim1, Ji-Cheon Kim1, Jang Won Yoon1, Kwang-Keun Oh2,
Jung-Ho Lee3, and Young Gyu Chai1*
1Division of Molecular and Life Sciences, Hanyang University, Ansan 426-791, Korea
2Department of Bioprocess Technology, BioPolytechnic College, Nonsan 320-905, Korea
3Department of Chemical Engineering, Hanyang University, Ansan 426-791, Korea
Received: June 16, 2007 / Accepted: September 4, 2007
Anthrax is an infectious disease caused by toxigenic
strains of the Gram-positive bacterium Bacillus anthracis.
To identify the mitochondrial proteins that are expressed
differently in murine macrophages infected with spores of
B. anthracis Sterne, proteomic and MALDI-TOF/MS
analyses of uninfected and infected macrophages were
conducted. As a result, 13 mitochondrial proteins with
different expression patterns were discovered in the infected
murine macrophages, and some were identified as ATP5b,
NIAP-5, ras-related GTP binding protein B isoform CRAa,
along with several unnamed proteins. Among these proteins,
ATP5b is related to energy production and cytoskeletal
rearrangement, whereas NIAP-5 causes apoptosis of host
cells due to binding with caspase-9. Therefore, this paper
focused on ATP5b, which was found to be downregulated
following infection. The downregulated ATP5b also reduced
ATP production in the murine macrophages infected with
B. anthracis spores. Consequently, this study represents
the first mitochondrial proteome analysis of infected
macrophages.
Keywords: Bacillus anthracis spores, mass spectrometry,
mitochondrial proteomics, murine macrophages
Bacillus anthracis is a Gram-positive, aerobic, rod-shaped
bacterium that forms endospores [8, 13] and interacts with
macrophages at various stages of infection. Moreover, anthrax
is a zoonosis to which most mammalian cells are susceptible
[20]. In the early stages of systemic anthrax, B. anthracis
spores interact with macrophages at the initial entry site into
the host. Once phagocytosed, endospores are simultaneously
germinated inside the macrophages and transported to the
regional lymph nodes [5, 6]. The germinated spores then
transform into vegetative bacilli and are able to grow through
replication under the conditions of the host intracellular
phagolysosome. Finally, the B. anthracis vegetative cells
are released from the phagocyte in order to spread [3].
To determine the relationship between the host and B.
anthracis interactions, a variety of research methods,
including proteomics and genomics, have already been
carried out. Nonetheless, there is still little evidence regarding
the necessary infection conditions for B. anthracis spores
in macrophages with regard to the interaction factor, cause
of host death, and immune response. As the host-pathogen
interaction can be better understood through the use of
proteomic and genomic techniques, a proteomic analysis
between macrophages and B. anthracis spores using two-
dimensional electrophoresis previously revealed protein changes
within macrophages with B. anthracis spores. Moreover,
various proteins, such as Pak2, related to the infection of B.
anthracis spores in macrophages, have been revealed in
the early stages [18]. A SELDI-TOF analysis was also recently
carried out to analyze the macrophage and B. anthracis spore
interaction and search for differentially expressed proteins
in macrophages during infection by B. anthracis [17].
Mitochondria are eukaryotic cellular organelles that are
important for cell death and known to be targeted for
infection by pathogenic bacteria. For example, pathogenic
bacteria and toxins are known to localize and induce
changes consistent with the permeabilization of mitochondria
[4, 22-24].
Accordingly, this study examined the mitochondria of
macrophages infected with B. anthracis Sterne spores, and
the results provide potential avenues for protection against
and therapy for the infection of B. anthracis spores in
macrophages.
*Corresponding author
Phone: 82-31-400-5513; Fax: 82-31-406-6316;
E-mail: ygchai@hanyang.ac.kr

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ATP SYNTHASE ACTIVITY BY B. ANTHRACIS SPORES
779
MATERIALS AND METHODS
Materials
The urea, thiourea, 3-[(3-cholamidopropy) dimethyammonio]-1-
propanesulfonate (CHAPS), 1% dithiothreitol (DTT), benzamidine,
Bradford solution, acrylamide, iodoacetamide, bis-acrylamide,
sodium-dodecyl sulfate (SDS), acetonitrile, trifluoroacetic acid, and
α-cyano-4-hydroxycinnamic acid were all purchased from Sigma-
Aldrich (St. Louis, MO, U.S.A.). The pharmalyte (pH 5-8) was
obtained from Amersham Biosciences (Piscataway, NJ, U.S.A.), the
immobilized IPGStrips (pH 5-8, 7cm) were from Bio-Rad (Hercules,
CA, U.S.A.), and the modified porcine trypsin (sequencing grade)
was from Promega (Madison, WI, U.S.A.).
Bacterial Strain and Mammalian Cell Culture
The B. anthracis strain used was Sterne (34F2; pXO1+, pXO2-) and
grown in a brain-heart infusion medium (BD Science, Ontario,
Canada) at 37oC. The total DNA was isolated according to a
previously reported method [17]. The Sterne strain was confirmed
by a PCR and used in spore formation. The murine macrophages
(macrophage-like RAW 264.7 cells) were maintained in Dulbecco’s
minimal essential medium (DMEM) supplemented with 10% FBS
(Gibco, Carlsbad, CA, U.S.A.), and then incubated at 37oC in an
atmosphere of 5% CO2 and saturating humidity.
Production of Bacillus anthracis Endospores
The B. anthracis spores were isolated and purified using a previously
reported method [3]. The vegetative cells were then inoculated onto
an NBY agar plate, and the cultures incubated at 37oC for seven days
until spore formation. The spores were confirmed by spore staining
and a PCR. The spores were collected from the plate and isolated
using sucrose gradient centrifugation. For nonsucrose solutions, the
top 9ml was transferred to an empty tube. The samples were then
centrifuged at 145×g for 30min and the supernatant was discarded.
The spores in the pellet were removed using sterilized distilled water,
and the pellet was resuspended in 5ml of PBS and stored at -20oC.
Infection of Murine Macrophages by Bacillus anthracis Sterne
Spores
The murine macrophages were infected with the B. anthracis spores
according to a previously reported method [3]. The murine
macrophages (1.34×107cells) were cultured in a culture plate
(Corning, Big Flats, NY, U.S.A.) at 37oC in 5% CO2 for 1 day. The
Sterne spores (1.36×108cells) were then inoculated at 37oC, and
transferred 30 min later to DMEM lacking antibiotics.
Protein Sample Preparation
The cultured cell pellets were quickly washed in ice-cold PBS. The
mitochondria isolation was performed using a Mitochondria Isolation
Kit (Pierce, Rockford, IL, U.S.A.). The cell pellet was collected by
centrifugation of the harvested cell suspension in a 2.0-ml
microcentrifuge tube at 850 ×g for 2 min. The supernatant was then
removed and discarded, and Mitochondria Isolation Reagent A
(800µl) added. The cells were vortexed at a medium speed for 5sec,
and the incubated tube was placed on ice for exactly 2min. Thereafter,
10µl of Mitochondria Isolation Reagent B was added, and the sample
vortexed at the maximum speed for 5sec. The incubation tube was
then placed on ice for 5min, and vortexed at the maximum speed
every min. Next, a total of 800 µl of Mitochondria Isolation Reagent
C was added, and then the tube was inverted several times to mix and
placed in a centrifuge at 700×g for 10min at 4oC. The supernatant was
subsequently transferred to a new 2.0-ml tube and placed in a centrifuge
at 12,000 ×g for 15 min at 4oC. To obtain a more purified fraction of
mitochondria, with a >50% reduction of lysosomal and peroxisomal
contaminants, the sample was placed in a centrifuge at 3,000 ×g for
15min. The supernatant (cytosol fraction) was transferred to a new
tube, while 500µl of Mitochondria Isolation Reagent C was added to the
pellet containing the isolated mitochondria and centrifuged at 12,000×g
for 5min. This time the supernatant was discarded and the mitochondrial
pellets were lysed with 2% CHAPS in Tris-buffered saline (TBS; 25mM
Tris, 0.15 M NaCl; pH 7.2). After centrifugation at 15,000 ×g for
1h at 15oC, the insoluble material was discarded and the soluble fraction
used for two-dimensional polyacrylamide gel electrophoresis (2D
PAGE). The protein loading was normalized by a Bradford assay [1].
2D PAGE
Immobilized pH gradient dry strips were equilibrated for 16 h with
7 M urea, 2 M thiourea containing 2% CHAPS, 1% DTT, and 1%
pharmalyte, and then loaded with 50 µg of the sample. The isoelectric
focusing (IEF) was performed at 20oC using a Protean IEF cell
electrophoresis unit (Bio-Rad, U.S.A.) following the manufacturer’s
instructions. For the IEF, the voltage was linearly increased from
150 to 4,000 V for 2 h during the sample entry, followed by a
constant 4,000 V, with focusing complete after 20,000 Vh. Prior to
the second dimension, strips were incubated for 10 min in an
equilibration buffer (50 mM Tris-HCl, pH 6.8, containing 6 M urea,
2% SDS, and 30% glycerol), first with 1% DTT and then with
2.5% iodoacetamide. The equilibrated strips were then placed on
SDS-PAGE gels (7-11 cm, 12%). The SDS-PAGE was performed
using a Bio-Rad Mini-Protean 3 system (Bio-Rad, Hercules, CA,
U.S.A.) following the manufacturer’s instructions, and the SDS-
PAGE gels were run at 20oC at 15 mA. Thereafter, the 2D PAGE
gels were silver stained as described previously [12], with the
omission of the fixing and sensitization step with glutaraldehyde.
Image Analysis
A quantitative analysis of the digitized images was carried out using
PDQuest (version 7.0, Bio-Rad, Hercules, CA, U.S.A.) software
according to the protocols provided by the manufacturer. The quantity
of each spot was normalized by the total valid spot intensity. Protein
spots with a significant expression level were selected and compared
with the B. anthracis Sterne spore sample after infection.
Enzymatic Digestion of Protein In-Gel
The protein spots were enzymatically digested in-gel in a manner
similar to that previously described [9, 19] using modified porcine
trypsin. The gel pieces were washed with 50% acetonitrile to
remove the SDS, salt, and stain, dried to remove the solvent, and
then rehydrated with trypsin (8-10 ng/µl) and incubated for 8-10 h
at 37oC. The proteolytic reaction was terminated by the addition of
5µl of 0.5% trifluoroacetic acid. The tryptic peptides were recovered
by combining the aqueous phase from several gel piece extractions
with 50% aqueous acetonitrile. Thereafter, the concentration of the
peptide mixture was desalted using C18ZipTips (Millipore, Bedford,
MA, U.S.A.), and the peptides were eluted in 1-5 µl of acetonitrile.
An aliquot of this solution was mixed with an equal volume of a
saturated solution of α-cyano-4-hydroxycinnamic acid in 50% aqueous
acetonitrile, and 1 µl of the mixture was spotted onto a target plate.

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780Seo et al.
MALDI-TOF Analysis and Database Search
A protein analysis was performed using an Ettan MALDI-TOF
(Amersham Biosciences, Piscataway, NJ, U.S.A.). The peptides
were evaporated with an N2 laser at 337 nm, using a delayed
extraction approach, and then accelerated with a 20-kV injection
pulse for a time-of-flight analysis. Each spectrum was the cumulative
average of 300 laser shots. The search program Mascot, developed
by Matrix Science [http://www.matrixscience.com/cgi/search_form.
pl?FORMVER=2&SEARCH=PMF], was used for the protein
identification based on peptide mass fingerprinting. The spectra
were calibrated using trypsin autodigestion ion peaks m/z (842.510,
2211.1046) as the internal standards.
Western Blot Analysis
Ten µg of mitochondrial extracts was separated on a 12% SDS-
PAGE gel. The gels were then electroblotted onto Western S
membranes (Schleicher & Schuell Bioscience, Keene, NH, U.S.A.)
and immunodetection was achieved using chemiluminescence
(Pierce, Rockford, IL, U.S.A.).
ATP Measurements
The ATP measurements were performed using bioluminescence in a
luminometer (Turner Designs, Sunnyvale, CA, U.S.A.) using ATP
assay reagents from Sigma-Aldrich.
RESULTS
A proteomic analysis of murine macrophages infected with
B. anthracis spores was carried out based on 2D PAGE of
murine macrophage mitochondrial extracts. In addition, a
Fig. 1. Comparative proteomic profiles of mitochondrial proteins from macrophage infected with Bacillus anthracis Sterne spores.
The proteins (50 µg) were separated by 2D PAGE, pH 5-8, and 12% SDS-PAGE gels, and the stains were carried out by alkaline silver staining. The figures
display sectors of interest at pH 5-5.5 within the 2D electrophoresis. The position of the 4101 spot is indicated (arrows). The expression of 4101 (arrows)
was analyzed after 0, 1, 2, and 4 h, respectively. This spot was identified by MALDI-TOF MS/MS.
Table 1. List of identified proteins.
Number of
sample
Name of protein
Sequence coverage
of protein (%)
Molecular
mass (Da)
Match of
peptide
pI
Mascot
score
2003
4101
4102
5101
5102
5103
5301
5402
6001
6002
6501
6502
7203
Ras-related GTP binding B, isoform CRAa
ATP5b protein
NAIP-5 (neuronal apoptosis inhibitory protein 5)
Unnamed protein product
Unnamed protein product
Unnamed protein product
4932439K10Rik protein
Unnamed protein product
Unnamed protein product
Tropomyosin 2, beta
Cdk5rap2 protein
Proteasome activator subunit 2 isoform 2
Unnamed protein product
07
12
02
04
08
05
11
04
20
09
02
06
05
040,542
056,632
161,871
086,776
042,039
029,972
088,220
068,673
016,250
032,933
157,161
026,317
056,249
3
7
3
3
3
2
8
2
4
4
4
2
3
5.38
5.24
5.58
5.1
5.29
5.46
5.20
5.42
28
90
30
31
39
36
44
36
34
55
28
35
28
4.66
5.17
5.08
6.42

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ATP SYNTHASE ACTIVITY BY B. ANTHRACIS SPORES
781
Western blot was performed on the mitochondrial extracts
to confirm the absence of lysosomal and peroxisomal
contaminants. No lysosomal or peroxisomal contaminants
were detected (data not shown). For the 2D PAGE, 50 µg
of the mitochondrial proteins was loaded on pH 5-8 IPG
strips. Alkaline silver staining was then used, which detected
about 200±20 spots per gel. Partial images are shown for
pH 5-5.5 (Fig. 1). The differential spots were analyzed
using PDQuest 7.0 image software. Seventeen spots of up-
/downregulated proteins were obtained (Table 1), among
which 13 proteins were identified and 4 proteins were not.
In particular, spot 4101 was monitored as a downregulated
protein (Fig. 2A), which was reduced by more than 50%
1 h after infecting the murine macrophages with the B.
anthracis Sterne spores, and reduced by about 75% after 4 h
(Fig. 2B). The ATP5b protein was identified by MALDI-
TOF/MS. A chromatogram of the ATP5b protein in the
mitochondria is represented in Fig. 3, which also shows the
coverage of the ATP5b protein (Fig. 3A). The MALDI-
TOF/MS spectrum of the trypsin-digested peptides derived
from the spot matched the ATP5b protein (Fig. 3B). The
patterns of the measured masses were matched against
the theoretical masses of proteins found in the annotated
database Mascot (http://www.matrixscience.com/cgi/search_
form.pl? FORMVER=2&SEARCH=PMF). The ATP5b
protein was identified by the Mascot database with a
Mowse Score of 90.
A Western blot analysis confirmed a continued decrease
in the ATP5b protein after 2 and 4 h following infection
with the B. anthracis Sterne spores (Fig. 4A). Meanwhile,
the total amount of ATP was analyzed by a bioluminescent
assay kit using a luminometer. Although the ATP amount
decreased after 1 h following the infection of the murine
macrophages with the B. anthracis Sterne spores (Fig. 4B),
Fig. 2. Comparison of mitochondrial protein expression patterns
of murine macrophage cells infected with Bacillus anthracis
Sterne spores from 0 to 4 h following infection.
A. Expression of protein indicated at spot 4101. B. Quantities of spot 4101
during B. anthracis spore infection of murine macrophage cells. Spot 4101
that appeared in the 2D PAGE gels was quantitatively analyzed using the
PDQuest analysis program.
Fig. 3. Peptide mass fingerprinting (A) and protein coverage map of ATP5b protein (B).
This protein was predicted by peptide fragments corresponding to the observed m/z values for ATP5b.

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782 Seo et al.
the ATP levels after 2 and 4 h were above the levels seen at
1 h after infection.
DISCUSSION
This study successfully used 2D PAGE to assess the
infection of murine macrophages with spores of B. anthracis.
Although the effects of B. anthracis spores on macrophages
are not yet clear, mitochondria are known to be the main
target of various intracellular pathogens. Thus, to examine
the protein changes within infected macrophages, proteomic
techniques were used to identify up- and downregulated
proteins within the mitochondria (Fig. 1). A novel approach
was also adopted to analyze the effects of infection with B.
anthracis Sterne spores using organelle proteomics.
Previous studies of mitochondria revealed several
pathogenic bacteria as cellular targets for mitochondria,
including Helicobacter pylori, Neisseria gonorrhoeae, and
Staphylococcus aureus [4, 22-24].
The present study used 2D PAGE to examine the
mitochondrial protein expression in macrophages infected
with B. anthracis. Thirteen among about 200 mitochondrial
proteins were observed to have a significant differential
expression after infection. These proteins were identified
by MALDI-TOF/MS and the Mascot database. Thus, based
on a good spectrum and high score obtained from the
Mascot database, this study focused on the ATP5b protein
that is related to mitochondrial ATP synthesis. Previous
studies of the ATP5b protein have reported that macrophages
infected with Francisella tularensis upregulated the ATP5b
protein [10], whereas macrophages exposed to the lethal
toxins of B. anthracis showed a reduction of the ATP5b
protein [2]. Recently, Sapra et al. [11, 16] reported that the
ATP5b protein was upregulated when a low concentration
of a lethal toxin was used to treat murine macrophages.
Additionally, Sapra et al. [16] reported an elevation of
cytoskeletal proteins and chaperones, such as Hsp70 and
Hsp60, in J774.1 and RAW264.7 macrophages exposed to
a lethal toxin. These chaperones are known to be involved
in protein folding and unfolding, and targeting irreversibly
denatured proteins for clearance. In addition, Hsp70 and
Hsp60 are both needed for binding ATP to protect against
apoptosis [14, 15, 21]. Cytoskeletal rearrangement also
needs ATP. Thus, the upregulation of ATP synthase in
lethal-toxin-treated macrophages may be generally correlated
to the production of ATP. However, Chandra et al. [2] reported
the downregulation of ATP and reduction of chaperones,
such as Hsp70, inducing cell death for macrophages.
Yet, this discrepancy may have resulted from the use of
different protocols for the experiments performed by the
different groups. Meanwhile, the present results indicated that
the ATP5b protein was downregulated in the macrophages
infected with B. anthracis (Fig. 2). This reduction of
ATP5b was also confirmed using a Western blot (Fig. 4A),
and the reduction of ATP confirmed by an ATP assay (Fig. 4B).
However, after 3 h, the amount of ATP was found to have
increased, while the amount of ATPase was decreased. In
mycobacteria, the acidification of the host cells is protected
by blocking the accumulation of the vacuolar proton
ATPase [8], which may explain the decrease in ATPase
during the early stage of Bacillus anthracis infection.
Nonetheless, Bacillus anthracis is dependent on the ATP
from the host cell for survival and proliferation. Similarly,
Chlamydia are entirely dependent on the ATP from the
host cells, so even if the expression of ATP5b decreases,
the amount of ATP increases [8]. Another possible reason
was that the macrophages infected with B. anthracis were
unable to generate cytoskeletal rearrangement and chaperones,
and thus the functions of the chaperones in the macrophages
were eliminated because of ATP depletion, thereby inducing
cell death. However, the cytoskeleton rearrangement and
expression of chaperone proteins were not confirmed in
this study. Therefore, it was assumed that the ATP5b
protein expression change was related to the use of the
host cell system and death of the host after infection
with B. anthracis. Although several previous reports have
demonstrated the differential mitochondrial protein expression
of macrophages infected with B. anthracis spores using
Fig. 4. Comparison of protein expression patterns of ATP5b
protein. A. Expression patterns of ATP5b protein using 2D-
PAGE and Western blot analysis. B. ATP assays of murine
macrophage cells infected with Bacillus anthracis Sterne spores.
The ATP5b protein was confirmed by a Western blot analysis. The
mitochondrial proteins were loaded with 10 µg of proteins. The ATP assay
was carried out in three independent experiments with the standard
deviation (SD) value depicted as an error bar.