Extracellular and Cytosolic Iron Superoxide Dismutase from Mycobacterium bovis BCG

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Extracellular and Cytosolic Iron Superoxide Dismutase from

Mycobacterium bovis BCG

SUNG-KOO KANG,

_{YONG-JAE JUNG,}

_{CHEORL-HO KIM,}

_{CHUL-YONG SONG}

_*

Department of Biology, Chung-Ang University, Dongjak-ku, Seoul 156-756,

_{and College of Oriental Medicine,}

Dongguk University, Kyoungju City, Kyungpook 780-714,

_Korea

Received 6 March 1998/Returned for modification 24 June 1998/Accepted 1 September 1998

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

approx-imately 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 H

O

, 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

2

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.

Superoxide dismutases (SODs), which catalytically scavenge

the superoxide radical (O2

_{) to hydrogen peroxide and}

mo-lecular 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

eu-karyotes, 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

num-ber of bacteria (10, 18).

Until now, numerous papers have reported the distribution,

characterization, and biological significance of SODs in

myco-bacteria (17, 19, 23). However, only a few myco-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

aster-oides GUH-2 secretes the enzyme into the growth medium (7).

SODs could also be involved in the pathogenicity of

Mycobac-terium leprae (33), although the secretion of the enzyme has

not been demonstrated and remains controversial.

Members of the mycobacteria have emerged as major

op-portunistic 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

dis-played 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

vacci-nation may cause disseminated mycobacterial infection, an

ill-ness 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.

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 What-man filter paper, washed three times with phosphate-buffered saline, and sus-pended 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-mm-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

220°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 50ml of 0.5 M potassium phosphate (pH 7.5), 25ml of 16% Triton X-100, 2.5ml of 10 mM EDTA, 75ml of 1.2 mM NTC, 2.5ml of xanthine oxidase (1.0 U), the sample, 25

ml of 4 mM hypoxanthine, and distilled water. The A540was monitored (Shi-madzu 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 ly-ophilized 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.

* 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

@cau.ac.kr.

784

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SDS-PAGE and native electrophoresis.Sodium dodecyl sulfate-polyacrylam-ide 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 (1mg) was preincubated with 3 mM cyanide, 20 mM azide, and 1 mM H2O2for 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 20mg of the purified enzyme was applied to an SDS-PAGE gel. After electrophoresis, the protein was electrophoretically transferred to a polyvinylidene difluoride mem-brane. 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 previ-ously (34). The sera used were from tuberculosis patients and were obtained from the Korea Institute of Tuberculosis.

RESULTS

Purification and properties of CE and CF SODs.

The SODs

were effectively purified by 70 to 90% ammonium sulfate

pre-cipitation and underwent hydrophobic interaction

chromatog-raphy with phenyl Sepharose CL-6B and Sephacryl S-200 gel

filtration. The results showed the SDS-PAGE pattern of

puri-fied CE SOD (20

m

g) and CF SOD (20

m

g) (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

2

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

electro-phoresis (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.

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.

The pI value of both enzymes was 4.5, and those areas of

activity were visualized in the same bands by zymography (Fig.

4).

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 (20mg); 2, purified CFSOD (20mg). Molecular weight (shown in parentheses) standard markers included the following proteins (Pharmacia): phosphorylase b (94,000), bovine serum albumin (67,000), ovalbu-min (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), anda -lact-albumin (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 aldo-rase (158,000) and bovine serum albumin (66,000).

TABLE 2. Purification of SOD from CFs of M. bovis BCG

Purification step proteinTotal (mg)

Total activity

(U)a

Sp act

(U/mg) purificationFold Recovery%

Crude extract

_10.08

_1,527.1

_141.4

_1.00

_100.0

HIC

_0.21

_933.3

_3,794.0

_26.83

_60.1

Gel filtration

_0.11

_444.4

_4,040.0

_28.57

_29.1

a_{A unit is defined as the amount of SOD needed to cause a 50% inhibition in}

the rate of oxidation of neotetrazolium chloride at 37°C.

b_{Seventy to 90% ammonium sulfate precipitation.} c_{Sepharose CL-6B hydrophobic interaction chromatography.} d_{Sephacryl S-200 superfine molecular sieve chromatography.}

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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

molec-ular weight of the standards yielded a subunit with a molecmolec-ular

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

FIG. 2. Molecular weight determination of purified SODs. (a) Molecular weight determination by SDS-PAGE (12.5% polyacrylamide). The marker pro-teins 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.

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;E_{, CF.}

FIG. 4. Isoelectric focusing analysis of purified enzymes. (a) Isoelectric fo-cusing 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 myo-globin acidic band (6.85), human carbonic anhydrase B (6.55), bovine carbonic anhydrase B (5.85),b-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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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.

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

pre-cipitated 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).

N-terminal amino acid sequence comparison.

When

puri-fied enzymes were subjected to Edman degradation and

deter-mination 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

iden-tical to that of CF SOD, implying that there are no N-terminal

signal peptides responsible for secretion. Although an

uniden-tified sequence existed, it was predicted that the first X of CE

SOD is glutamic acid (E), as judged from Edman degradation

data.

Western blot analysis.

To examine whether the purified

en-zymes are working as antigenic molecules in tuberculosis

pa-tients, 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

indi-cated that both of the purified SODs have the same antigenic

determinant, as shown by antigenicity against the sera of

tu-berculosis patients.

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,

sub-unit 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

extracel-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 mercap-toethanol, 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.

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.

P. aeruginosa A---FELPPLPYEKNAL Hassett et al. (14) B. fragilis (GenBank

accession no. M96560)

TYEMPKLPYANNAL Unpublished

observations (1992)

a_{X, undetected amino acid sequence. Boldface indicates identical amino}

ac-ids.

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lular 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

Esch-erichia coli (60%), Pseudomonas aeruginosa (40%), and

Bacte-roides fragilis (33%) (Table 3). The correspondence in

N-ter-minal 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

secre-tion 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

exporta-tion exist in mycobacteria. Further investigaexporta-tion 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

z

), a highly reactive molecule that will

react with various biomolecules, including lipids, proteins, and

DNA, both by reducing Fe

_{to Fe}

_{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

super-oxide 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

anti-genic determinant. This result showed that this SOD could be

used as a marker protein for diagnosis of tuberculosis.

How-ever, 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.

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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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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