Clinical and Diagnostic Laboratory Immunology, November 1998, p. 784-789, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Extracellular and Cytosolic Iron Superoxide
Dismutase from Mycobacterium bovis BCG
Sung-Koo
Kang,1
Yong-Jae
Jung,1
Cheorl-Ho
Kim,2 and
Chul-Yong
Song1,*
Department of Biology, Chung-Ang University,
Dongjak-ku, Seoul 156-756,1 and
College
of Oriental Medicine, Dongguk University, Kyoungju City, Kyungpook
780-714,2 Korea
Received 6 March 1998/Returned for modification 24 June
1998/Accepted 1 September 1998
 |
ABSTRACT |
Two forms of iron superoxide dismutase (SOD) were purified from
cell extract (CE) and culture filtrate (CF) of Mycobacterium bovis BCG, respectively. The molecular weight of both enzymes was
estimated to be approximately 84,000 by gel filtration, whereas that of
their subunits was 21,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that each of
purified enzymes is composed of four identical subunits. The specific
activities of CE SOD and CF SOD were 3,850 and 4,040, respectively. The
purified enzymes were not joined by disulfide bonds and were, to some
extent, resistant to sodium dodecyl sulfate. Their activities were lost
by H2O2, but not by azide and cyanide, indicating iron SODs. Enzyme activities were detectable over a broad
range of pHs, from 5.0 to 9.0, and were stable for 6 months at
20°C. Each value of pI was 4.5. In Western blots, both enzymes reacted with sera of tuberculosis patients, but not with normal sera.
The N-terminal amino acid sequences of CE SOD and CF SOD were the same,
suggesting that there is no N-terminal signal sequence.
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INTRODUCTION |
Superoxide dismutases (SODs), which
catalytically scavenge the superoxide radical
(O2
) to hydrogen peroxide and molecular
oxygen, serve a protective role against oxygen toxicity in all aerobic
organisms (13, 37). Because the superoxide radical is a
normal product of the univalent reduction of molecular oxygen, SODs are
thought to be the primary defense against its potential cytotoxicity
(13). There are three common forms of SODs, which differ in
the metal ion cofactor at the active site. Manganese-containing SODs
(MnSODs) are found in bacteria and mitochondria, while iron-containing
SODs (FeSODs) are mainly found in the cytosol of prokaryotes, in
primitive eukaryotes, and in some green plants (4, 5). In
contrast, copper-zinc SODs (Cu-ZnSODs) are mostly found in the cytosol
of eukaryotes, surprisingly in an increasing although limited number of
bacteria (10, 18).
Until now, numerous papers have reported the distribution,
characterization, and biological significance of SODs in mycobacteria (17, 19, 23). However, only a few bacteria have been shown to secrete SOD during growth. It was shown previously that
Mycobacterium tuberculosis, a human pathogen, secreted iron
SOD, while the nonpathogenic species, Mycobacterium
smegmatis and Mycobacterium phlei, did not
(19). Similarly, it was shown previously that the virulent strain Nocardia asteroides GUH-2 secretes the enzyme into
the growth medium (7). SODs could also be involved in the
pathogenicity of Mycobacterium leprae (33),
although the secretion of the enzyme has not been demonstrated and
remains controversial.
Members of the mycobacteria have emerged as major opportunistic
pathogens in humans, with the advent of the AIDS epidemic (16). Recently, such a case was also reported in a human
immunodeficiency virus-infected individual who displayed reactivation
of Mycobacterium bovis BCG 30 years after vaccination
(3), although M. bovis BCG has been used in many
countries for vaccination to prevent tuberculosis. BCG vaccination may
cause disseminated mycobacterial infection, an illness caused by the
vaccine itself in patients suffering from severe immune deficiency.
In this study, we explored the production and secretion of SOD by
M. bovis BCG by investigating the purification and some properties of M. bovis BCG SOD along with evidence of the
association of this enzyme with the cytosol and the growth medium of
the organism.
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MATERIALS AND METHODS |
Bacterial strain and culture condition.
The M. bovis BCG strain used in this study was Pasteur strain 1173P2 and
was obtained from the Korea Institute of Tuberculosis. The bacterium
was cultured at 37°C in Sauton's medium (30) without Triton.
Preparation and fractionation of CEs and CFs.
Cells were
collected on Whatman filter paper, washed three times with
phosphate-buffered saline, and suspended with sterilized distilled
water. After homogenization, cells were disrupted with a French press
(Aminco, Rochester, N.Y.) at a pressure of 18,000 lb/in2
and centrifuged at 15,000 rpm for 30 min at 4°C. Culture filtrates (CFs) were filtered through a 0.2-µm-pore-diameter membrane (Gelman, Ann Arbor, Mich.) and concentrated by an Amicon concentrator with a
YM-10 membrane (Amicon, Beverly, Mass.). Each of the cell extracts (CEs) and CFs was precipitated by 70 to 90% ammonium sulfate, suspended with D.W., lyophilized, and stored at 20°C.
Enzyme assay and protein determination.
The activity of the
enzyme was assayed at 37°C for 30 min. The assay mixtures (0.5 ml)
contained 50 µl of 0.5 M potassium phosphate (pH 7.5), 25 µl of
16% Triton X-100, 2.5 µl of 10 mM EDTA, 75 µl of 1.2 mM NTC, 2.5 µl of xanthine oxidase (1.0 U), the sample, 25 µl of 4 mM
hypoxanthine, and distilled water. The A540 was
monitored (Shimadzu UV-240) after addition of 0.5 ml of a solution
containing 1 M formate buffer (pH 3.5), 10% Triton X-100, and 40%
formaldehyde. An enzymatic unit was defined as the amount of the enzyme
required to cause a 50% inhibition in the rate of reduction of NTC
under the assay conditions (27). The standard protein used
in unit determination was SOD from bovine kidney (Sigma, St. Louis,
Mo.).
The protein concentration was determined by the Lowry method
(21) with bovine serum albumin as a standard.
Purification of CE SOD and CF SOD.
Crude CE and CF extracts
were dialyzed against 50 mM Tris-HCl (pH 8.0) containing 0.5 M ammonium
sulfate, briefly centrifuged, and chromatographed on phenyl Sepharose
4B (1.6 by 15 cm [Sigma]) at room temperature in the same buffer. The
gradient procedure was performed with D.W. at a flow rate of 30 ml/h.
SOD fractions were dialyzed against D.W. (pH 7.0) and lyophilized
(Labconco, Kansas City, Mo.). The lyophilized material was dissolved
with 50 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl prior to
chromatography at a flow rate of 20 ml/h on a Sephacryl S-200
(Pharmacia, Uppsala, Sweden) gel filtration (1.6 by 85 cm) system
calibrated with standard proteins (Pharmacia) at 4°C.
SDS-PAGE and native electrophoresis.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as
described previously (20) on a minigel comprising a 12.5%
acrylamide continuous resolving gel, and native electrophoresis was
carried out by the method of Bollag and Edelstein (11).
Enzyme activity staining following native electrophoresis.
SOD activity was visualized on a polyacrylamide gel as described
previously (9). The SOD-active area appeared as a clear zone
on a blue-violet background.
Inhibition test.
To test inhibition, each SOD (1 µg) was
preincubated with 3 mM cyanide, 20 mM azide, and 1 mM
H2O2 for 30 min and electrophoresed on 12.5%
polyacrylamide gel prior to enzyme activity staining.
Enzyme stability and isoelectric focusing.
After 12.5%
polyacrylamide native gel electrophoresis, each lane was soaked for
12 h at pH 4.5 to 5.0 (sodium acetate buffer), 6.0 to 7.5 (sodium
phosphate), 8.0 to 8.5 (Tris-HCl), and 9.0 (glycine-NaOH), and enzyme
activity staining was carried out before the results were read at 550 nm in a densitometer (LKB, Bromma, Sweden). Isoelectric focusing was
carried out automatically with Phastsystem (Pharmacia, Sweden).
N-terminal sequencing of the purified SOD.
Approximately 20 µg of the purified enzyme was applied to an SDS-PAGE gel. After
electrophoresis, the protein was electrophoretically transferred to a
polyvinylidene difluoride membrane. The area containing the band was
cut out and subjected to N-terminal amino acid sequence analysis with a
protein sequencer (Milligen 6600B).
Western blot analysis.
Western blotting was carried out as
described previously (34). The sera used were from
tuberculosis patients and were obtained from the Korea Institute of Tuberculosis.
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RESULTS |
Purification and properties of CE and CF SODs.
The SODs were
effectively purified by 70 to 90% ammonium sulfate precipitation and
underwent hydrophobic interaction chromatography with phenyl Sepharose
CL-6B and Sephacryl S-200 gel filtration. The results showed the
SDS-PAGE pattern of purified CE SOD (20 µg) and CF SOD (20 µg)
(Fig. 1a). A single band with a molecular weight of about 21,500 was stained by Coomassie brilliant blue. The
specific activity of CE-SOD was 3,850 U/mg of protein, representing a
purification of 30.1-fold with 53.5% recovery. CF SOD was purified approximately 28.57-fold with a specific activity of 4,040 U/mg of
protein and 29.1% recovery (Tables 1 and
2). The activities of purified enzymes
were stable at 20°C for 6 months (data not shown). The molecular
weights of the native enzymes, as determined by gel filtration on
Sephacryl S-200 (Fig. 2b) and native
electrophoresis (Fig. 1b), were about 84,000. These results indicated
that each of the purified SODs is a tetramer composed of identical
polypeptides with a molecular weight of about 21,500.
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FIG. 1.
SDS-PAGE and native electrophoresis analysis of purified
enzymes. (a) SDS-PAGE (12.5% polyacrylamide) pattern. Lanes: M,
standard marker proteins; 1, purified CE SOD (20 µg); 2, purified
CFSOD (20 µg). Molecular weight (shown in parentheses) standard
markers included the following proteins (Pharmacia): phosphorylase
b (94,000), bovine serum albumin (67,000), ovalbumin
(43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and
 -lactalbumin (14,400). (b) Native (12.5% polyacrylamide)
electrophoresis pattern. Lanes: M, standard marker proteins; 2, purified CE SOD; 4, purified CF SOD; 1 and 3, no protein. Standard
marker proteins (Pharmacia, Sweden) were aldorase (158,000) and bovine
serum albumin (66,000).
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FIG. 2.
Molecular weight determination of purified SODs. (a)
Molecular weight determination by SDS-PAGE (12.5% polyacrylamide). The
marker proteins were the same as in Fig. 1a. (b) Molecular weight
determination by Sephacryl S-200 molecular sieve chromatography. The
molecular weight (shown in parentheses) marker proteins (Pharmacia)
were as follows: aldorase (158,000), bovine serum albumin (67,000),
ovalbumin (43,000), chymotrypsinogen A (25,000), and RNase A (13,700).
Arrows indicated the purified SODs.
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The stability of both enzymes was detectable over a broad range of pHs,
5.0 to 9.0, but CF SOD was a little more stable than CE SOD at pH 4.0 (Fig. 3). For unknown reasons, the
specific activity and stability of CF SOD are higher than those of CE
SOD.
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FIG. 3.
Effect of stability of pH on the activity of purified
enzymes. After native gel (12.5% polyacrylamide) electrophoresis,
sliced gels were soaked in various pH buffers (pH 4.0 to 9.0) at 37°C
for 12 h, incubated in staining solution containing 0.25 mM nitro
blue tetrazolium for 30 min at 37°C, and read at 550 nm in a
densitometer (LKB). Maximal activity was determined as 100%. The
buffers used were 0.1 M sodium acetate (pH 4.0 to 5.0), 0.1 M sodium
phosphate (pH 6.0 to 7.5), 0.1 M Tris-HCl (pH 8.0 to 8.5), and 0.1 M
glycine-NaOH (pH 9.0).  , CE;  , CF.
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The pI value of both enzymes was 4.5, and those areas of activity were
visualized in the same bands by zymography (Fig.
4).
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FIG. 4.
Isoelectric focusing analysis of purified enzymes. (a)
Isoelectric focusing in 3-9 IEF premade gel (Pharmacia). (b) Activity
staining. Lanes: M, standard markers; 1, purified CE SOD; 2, purified
CF SOD. Isoelectric point (shown in parentheses) markers included the
following proteins (Pharmacia): trypsinogen (9.30), lentil lectin basic
band (8.65), lentil lectin middle band (8.45), lentil lectin acidic
band (8.15), horse myoglobin basic band (7.35), horse myoglobin acidic
band (6.85), human carbonic anhydrase B (6.55), bovine carbonic
anhydrase B (5.85),  -lactoglobin (5.20), soybean trypsin inhibitor
(4.55), and amyloglucosidase (3.50). Note that not all marker positions
are labeled on the figure.
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Subunit molecular weight.
The enzymes preincubated with sample
buffer for 2 h at 37°C in the presence and absence of
2-mercaptoethanol in sample buffer were subjected to SDS-PAGE (4,
37). Comparison of their mobility to that of molecular weight of
the standards yielded a subunit with a molecular mass of 21,500 Da in
both the absence and presence (Fig. 5) of
mercaptoethanol, indicating that the enzymes are not affected by
mercaptoethanol. Even though a 66,000-Da protein band was also detected
on the electrophoresis gel, it was suggested that the native enzyme
structures are resistant to SDS because of their partial disruption by
SDS treatment. From this result, it is clear that the enzymes are
composed of four subunits of equal size and that these subunits are not
joined by interchain disulfide bonds.
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FIG. 5.
Determination of subunit molecular weight of purified
enzymes by 12.5% acrylamide gel electrophoresis. Lanes: M, marker
proteins; 1 and 2, CE SOD; 3 and 4, CF SOD. Lanes 1 and 3 show results
in the absence of mercaptoethanol, and lanes 2 and 4 show results in
the presence of it. Each of the samples was incubated at 37°C for
2 h. The marker proteins used were the same as in Fig. 1a.
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Effect of inhibition.
In Fig. 6,
both of the purified CE and CF SODs were inhibited completely by 1 mM
H2O2 (CE and CF in lane 4), but not by 10 mM
azide (CE and CF in lane 3) and 3 mM cyanide (CE and CF in lane 2), as
shown by zymography. This indicated that the purified enzymes are
iron-SOD (4, 35) and that CF SOD may be a secreted form of
CE SOD. To confirm this hypothesis, when a 3-day culture filtrate was
precipitated by 85% ammonium sulfate and dialyzed with D.W., a protein
band with the same molecular weight of the purified CF SOD appeared by
activity-staining electrophoresis (data not shown).
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FIG. 6.
Effects of inhibitors on purified enzymes. One microgram
of the enzyme was preincubated with 3 mM cyanide (lane 2), 20 mM azide
(lane 3), and 1 mM H2O2 (lane 4), respectively,
for 30 min and electrophoresed on a 12.5% acrylamide gel prior to
enzyme activity staining. Lane 1 contained the control.
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N-terminal amino acid sequence comparison.
When purified
enzymes were subjected to Edman degradation and determination of
N-terminal amino acid sequences, the N-terminal amino acid sequence of
CE SOD was AXYTLPDLDX and that of CF SOD was AEYTLPDLDXDYGAL. Ten of
the N-terminal amino acid sequences in CE SOD and 15 of those in CF SOD
in this study showed a very high degree of similarity to those of other
mycobacterial species and N. asteroides and low homology
with those of other bacteria (Table 3).
It is interesting that the N-terminal sequence of CE SOD was identical
to that of CF SOD, implying that there are no N-terminal signal
peptides responsible for secretion. Although an unidentified sequence
existed, it was predicted that the first X of CE SOD is glutamic acid
(E), as judged from Edman degradation data.
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TABLE 3.
N-terminal sequence comparison of M. bovis BCG
SOD with SODs from other bacteria characterized in the NCBI
GenBank database
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Western blot analysis.
To examine whether the purified enzymes
are working as antigenic molecules in tuberculosis patients, Western
analysis was carried out with sera from the patients. Both CE SOD (Fig.
7, CE, lanes 1 to 8) and CF SOD (Fig. 7,
CF, lanes 1 to 4) were reacted with sera of tuberculosis patients, but
were not reacted with normal sera (Fig. 7, CE, lanes 9 to 12, and CF,
lanes 5 to 8). Even though only one result is presented, the Western
blotting data strongly indicated that both of the purified SODs have
the same antigenic determinant, as shown by antigenicity against the sera of tuberculosis patients.
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FIG. 7.
Western blot analysis of purified enzymes. SDS-PAGE
(12.5% polyacrylamide) and electrophoretic transfer of purified SOD
onto nitrocellulose membranes were performed as described in Materials
and Methods. Arrows indicate the purified SOD bands of CE (a) and CF
(b). Lanes 1 to 8 in panel a (CE) and 1 to 4 in panel b (CF) show the
reaction with sera from patients infected with M. tuberculosis; lanes 9 to 12 in panel a (CE) and 5 to 8 in panel b
(CF) show results with normal sera from healthy individuals.
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DISCUSSION |
Iron-SOD has been purified from CE and CF of M. bovis
BCG grown in Sauton's medium. Purified enzymes showed similarities
with respect to molecular weight, pH profile, subunit structure,
substrate specificity, sensitivity to inhibitors, and antigenicity to
tuberculosis patients. The molecular weight and tetrameric form of the
enzymes (Fig. 1) were similar to those of SODs of M. tuberculosis (19), Mycobacterium avium
(12), and N. asteroides (7). However, there is a slight difference from those of M. leprae,
M. smegmatis, Mycobacterium lepraemurium,
Mycobacterium intracellulare, and Mycobacterium
duvalii, even with BCG (22). Since adoption of a
tetrameric form is thought to confer the stability of the enzyme in
cellular and extracellular fluids (15), the purified SODs showed a broad pH stability (Fig. 3). These results were similar to
those with many other SODs (24, 28, 31).
CE SOD and CF SOD shared strong N-terminal amino acid sequence
homologies with SODs from mycobacteria, such as M. tuberculosis (93%), Mycobacterium fortuitum (93%),
M. avium (87%), M. leprae (87%), and N. asteroides (87%), but low homologies with those from other
bacteria, such as Escherichia coli (60%), Pseudomonas aeruginosa (40%), and Bacteroides fragilis (33%)
(Table 3). The correspondence in N-terminal amino acid sequence between
CE SOD and CF SOD indicated that the SOD of M. bovis BCG is
not preceded by signal peptides. Thus, it was suggested that the
structural gene of the enzyme from M. tuberculosis is not
preceded by a signal peptide sequence (38). Although
M. bovis BCG iron SOD appeared to be cytosolic as well as a
secreted protein, it was not the product of autolysis, because it was
found in CF within 3 days of growth under identical culture conditions.
SODs from CE and CF showed identical molecular weights, pIs, metal
cofactors, N-terminal amino acid sequences, and Western blots,
suggesting that CF SOD should be due to direct secretion from CE SOD.
How SOD is exported in mycobacteria remains unknown, since there are no
possible signal peptides (12, 38). Probably, some specific
systems for protein exportation exist in mycobacteria. Further
investigation is in progress to explain the secretion mechanism of the
enzyme in the outer cell surface of the bacterium and at the gene level.
It has been reported that superoxide enhances formation of hydroxyl
radical (OH·), a highly reactive molecule that will react with
various biomolecules, including lipids, proteins, and DNA, both by
reducing Fe3+ to Fe2+ and by serving as a
source of H2O2 (26). Most bacteria
contain SODs and catalase as means of eliminating superoxide and
H2O2, respectively. Pathogenic microorganisms
are exposed to exogenous superoxide and H2O2
generated by host neutrophils and other phagocytes (26). The
secreted SOD has been documented to occur in a few virulent
mycobacteria (2, 7, 12), showing that it could be involved
in pathogenicity (6, 12, 19). The secretion of SOD in
virulent forms of N. asteroides, as well as its association with the outer cell envelope, could provide protection against killing
by superoxide radicals (6), which are produced during active
phagocytosis. More direct evidence for such a protective role had been
obtained previously via administration of a monoclonal antibody
specific for the SOD of N. asteroides (8). The
fact that exogenously added SOD has protected bacteria against
phagocytic attack (36) also illustrates the importance of
SOD. Therefore, the SOD secreted by mycobacteria is more important,
because it could function in the bacteria as a first line of defense
against oxygen-mediated killing.
It has been also reported that the SOD in the purified protein
derivative of M. tuberculosis used for the skin test is a
general antigen (29). In this study (Fig. 7), both M. bovis BCG CE SOD and CF SOD obviously showed antigenicity against
sera from patients with tuberculosis and had the same antigenic
determinant. This result showed that this SOD could be used as a marker
protein for diagnosis of tuberculosis. However, the specificity and
sensitivity need to be determined with a larger number of sera from
patients with different stages of tuberculosis and other mycobacterial infections and from BCG-vaccinated or nonvaccinated individuals.
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ACKNOWLEDGMENTS |
We thank Yong-Gil Park of the Korea Institute of Tuberculosis for
providing the bacterial strain and patients' sera, Samuel Len of the
Korea Herald for proofreading the manuscript, and Chul-Sun Choi of Chung-Ang University for helpful review. This study was supported in part by a Special Research grant from Chung-Ang University.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Chung-Ang University, 221, hueksuk-dong, Dongjak-ku, Seoul
156-756, Korea. Phone: 82 02 820 5208. Fax: 82 02 816 6710. E-mail:
cysong{at}cau.ac.kr.
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Clinical and Diagnostic Laboratory Immunology, November 1998, p. 784-789, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
