l'eterinary Microbiology, 26 ( 1991 ) 359-366 Elsevier Science Publishers B.V., A m s t e r d a m
Superoxide dismutases of virulent and avirulent strains of Brucella abortus Nammalwar Sriranganathana, Stephen M. Boyle a, Gerhardt Schuriga and Hara Misra b
Departments of~Pathobiology and hBiomedical Sciences, Va-Md Regional College of Veterinary Medicine. Virginia Polytechnic Institute and State University Blacksburg, VA 24061, USA (Accepted 9 August 1990 )
ABSTRACT Sriranganathan, N., Boyle, S.M., Schurig, G. and Misra, H., 1991. Superoxide dismutases of virulent and avirulent strains ofBrucella abortus. Vet. Microbiol., 26: 359-366. Extracts of Brucella abortus strains 2308,RB51,45/20 and ST 19 had no significant differences in superoxide dismutase (SOD) activity as measured by the epinephrine assay. These B. abortus strains represent smooth, intermediate and rough colony forms. SOD activity was inhibited 60 to 75% by 2 mM KCN and suggests the presence of C u / Z n SOD. The SOD activities were similar when the strains were grown in trypticase soy broth containing either 0.5% glucose or erythritol. There were lwo distinct SOD activity bands in native polyacrylamide gel electrophoresis with identical mobilities for each of the strains. When the native gel was stained for SOD activities in the presence of 2 mM KCN, the SOD band that co-migrated with the bovine erythrocyte C u / Z n SOD activity disappeared. The band of SOD activity that migrated similar to E. coli iron SOD activity was unaffected by KCN. There were no significant differences in either the total SOD or C u / Z n SOD activities among the strains. As the Brucella strains represent ranges of virulence, it is difficult to associate any primary role for SOD as a virulence factor.
Superoxide dismutase (E.C. 126.96.36.199 ) (SOD) activity is present in all oxygen metabolizing cells and catalyzes the dismutation of toxic superoxide radicals into hydrogen peroxide and molecular oxygen (McCord et al., 1971 ). SOD was first purified from bovine erythrocytes by McCord and Fridovich (1969). Since then there have been a numbers of reports on the presence of SOD's in aerobically growing cells (Misra and Fridovich, 1972a; Misra and Keele, 1975; Misra and Fridovich, 1977; Britton et al., 1978; Moore et al., 1984). Superoxide radicals (O~-) are generated as intermediates during reduction of molecular oxygen. In addition, these oxygen radicals undergo further reduction to form hydrogen peroxide (H202) and the hydroxyl radical ( O H - ) (Ewing, 1983). These active oxygen species (O~-,H202, OH" ) can
© 1991 - - Elsevier Science Publishers B.V.
N. SRIRANGANATHANET AL.
damage DNA, RNA, proteins and lipids (Mead, 1976; Bielski and Shiue, 1979; Halliwell and Gutteridge, 1984; Storz et al., 1987) leading to disruption of cellular architecture and activity (Farr et al., 1988 ). To protect themselves against these toxic species, cells that can grow in presence of oxygen utilize antioxidant enzymes eg. SOD, catalases and peroxidases. SODs in bacteria play an important role which permit them to survive phagocytic attack. Thus, SOD may be regarded as a virulence factor facilitating intracellular survival (Wollinsky, 1979; Beaman et al., 1983; Farr et al., 1988). There are three distinct SODs that have been characterized on the basis of their metal prosthetic groups. These are copper/zinc, iron and manganese containing SODs (Fridovich, 1984). The C u / Z n SOD is mainly present in the cytosol of eukaryotes and is considered the most recently evolved SOD (Britton et al., 1978). Bacteria, like most prokaryotes produce either the Fe a n d / o r Mn SOD (Bannister and Rotillo, 1984). Exceptions to these generalizations are a Mn SOD, which has been isolated from the mitochondria of eukaryotes, and a C u / Z n SOD which has been identified in a few prokaryotes like Photobacterium leiognathi (Bannister and Parker, 1985; Steinman, 1987 ), Pseudomonas diminuta and Pseudomonas maltophilia (Steinman, 1985 ). Brucella are facultative intracellular bacterial pathogens which are able to reside and multiply within macrophages. The ability of Brucella to replicate within macrophages depends on their ability to inhibit a n d / o r resist intraleukocytic killing mechanisms. The presence of superoxide dismutase activity has been shown to be positively correlated with increased pathogenicity in a number of bacterial strains (Beaman et al., 1985; Kanafani and Martin, 1985 ). Recently, Beck et al. (1990) have isolated an envelope associated (high salt extractable ) protein from B. abortus and found that it is a C u / Z n SOD. They have shown that the Brucella C u / Z n SOD has high homology (53.6%) to Photobacterium leiognathi C u / Z n SOD. The present communication describes the presence of cyanide sensitive and cyanide insensitive forms of SODs in a vaccine strain of Brucella as well as other strains orB. abortus with different degrees of virulence. MATERIALS AND METHODS
Strains of B. abortus used in this study (Table 1 ) were from the culture collection of G. Schurig. Strain RB51 is a rough, rifampin resistant mutant derived from the virulent strain 2308 (Schurig et al., submitted). All four strains were cultured on trypticase soy agar (Baltimore Biological Laboratories, M D ) from stocks maintained at - 8 0 ° C. All strains were grown in 30 ml of TSB in 250 Erlenmeyer flasks at 37 °C in a shaker incubator using a t % inoculum of an overnight culture. The cells were grown in TSB containing either 0.5% glucose or erythritol. The cells were harvested at late log phase (Klett units = 350 at 540 nM) by centrifugation in
BRUCELLA SUPEROXIDE DISMUTASES
TABLE 1 Strains ofB. abortus used in the study Strain
2308 19 45/20 RB51
+ + + + + + + + +
Smooth Smooth Intermediate Rough
~Defined as colony forming units needed to achieve infection of mice for over 3 weeks. + + + = 10: to 104, + + = 104 to 106, + = 107 to 109 (Montaraz and Winter, 1986).
aerosol free centrifuge bottles. The cells were washed once in 50 mM potassium phosphate buffer (pH 7.8) and resuspended in 3 ml of the same buffer. The cells were sonicated at 4°C at a setting of 6 on the output control of a sonicator (Model W-225 Heat Systems-Ultrasonics, Inc) for 3 min using an intermittent pulsed (50%) duty cycle. The sonicate was centrifuged at 10 000 × g for 20 min. The supernatants were filter sterilized using a 0.2/~M nitrocellulose membrane filter (Millipore Corporation, MA USA). Approximately 3 ml of the crude extracts were aliquoted into 500/~1 samples and stored at - 80 °C until used. Purified Cu/Zn, Fe and Mn SODs were purchased from Sigma Chemical Co (St. Louis, MO ) were used as controls in the SOD assays. The SOD enzyme activity was measured by an epinephrine assay (Misra and Fridovich, 1972b). The amount of SOD required to inhibit the rate of auto-oxidation of epinephrine by 50% was defined as 1 unit of activity. The protein content was estimated colorimetrically (BioRad reagent, BioRad Richmond, CA) using bovine serum albumin as the standard. Extracts were electrophoresed at 30 mA in 10% native polyacrylamide gels ( 10 cm) using a 10% stacking gel (Gordon, 1971 ). SOD activity was detected using the nitroblue tetrazolium reduction method (Beauchamp and Fridovich, 1971 ). Two millimolar of either KCN or sodium azide was used to determine the inhibitory action on SOD activity in the enzyme assays as well as in native PAGE gels. RESULTS
The specific activities of SOD in crude extracts from cells grown in glucose or erythritol of the four B. abortus strains are shown in Table 2. Analysis of variance of the data showed that there was no significant difference in the SOD activity of cells grown in either glucose or erythritol. There was no detectable SOD activity in the culture supernatants. There was however, a tendency for all strains of Brucella to exhibit lower SOD specific activity when grown in erythritol compared to glucose. This may be due to a higher release
N. SRIRANGANATHANET AL.
TABLE 2 Specific activity of SOD from four strains ofB. abortus in the epinephrine assay B. abortus strains
2308 RB51 45/20 19 2308 RB51 45/20 19 ~
3 2 2 2 3 3 3 ND
Glu Glu Glu Glu Ery Ery EU"
Specific activity SOD (U/ mg-+ SD) 410.7 _+82 368.5_+20 488.2 + 30 609.9_+ 3 238.9_+93 296.5_+22 298.2_+90
Percent of total SOD Cu/Zn 2
64 75 61 76 68 67 69
36 25 39 24 32 33 31
~Strain 19 is unable to grow in presence of erythritol; N D = n o t determined. 2The cyanide sensitive portion (Cu/Zn) was subtracted from the total activity to derive cyanide insensitive (Mn(Fe) portion.
Fig. 1. Native polyacrylamide gel electrophoresis of crude extracts loaded in lanes from left to right. Gel A without cyanide, Gel B with 2 m M KCN. Both the gels A a n d B were loaded with identical a m o u n t s of the crude extracts. Lane 1 = 10 lzl of St 19 G (3.26 #g); lane 2 = 10/zl of 2308 G (2.76/tg); lane 3 = 10/zl of 4 5 / 2 0 G ( 1.21 pg); lane 4 = 10pl o f R B 5 1 G (3.44 izg); lane 5 = 0 . 7 5 U of C u Z n SOD from b o v i n e erythrocyte standard; lane 6 = 0 . 7 5 U of Fe SOD from E. coli; lane 7 = 0 . 7 5 U of M n SOD from E. coli; lane 8 = 10/~1 of 2308 E (9.82 pg): lane 9--- 10~tt of 4 5 / 2 0 E (9.49~tg); lane 1 0 = 10~tl o f R B 5 1 E (9.54,ug).
BRUCELLA SUPEROXIDE DISMUTASES
of protein by sonication from the cells grown in erythritol (protein concentration of crude extract 1.17 +_0.02 m g / m l ) compared to cells grown in glucose (0.63 + 0.14 m g / m l ) . In the epinephrine assay, the percentage of C u / Z n SOD was approximately 64 to 75% of the total SOD in all Brucella strains tested. When the crude extracts were electrophoresed in native PAGE gels, the samples produced two distinct bands (R.F. 0.39 and 0.66) upon activity staining. Scanning densitometry revealed that the upper band had approximately 65% of the activity compared to the 35% for the lower band (Fig. 1, Gel A). These values closely agreed with those obtained in the epinephrine assay. When a duplicate gel was treated with 2 m M KCN during staining, the upper band and the bovine C u / Z n SOD standard disappeared. The lower band showed some loss of staining intensity (Fig. 1, Gel B). With strain 19, there was an additional lower band of activity (Fig. 1, Gel A and B) that was cyanide insensitive. This band disappeared when a smaller amount of protein was loaded suggesting that the band of activity may be due to dissociation. When 5 m M sodium azide was used as inhibitor in similar gels there was no appreciable loss of activity of either bands. Similar concentrations of azide inhibited the E. coli Fe SOD 50 to 60% in the epinephrine assay when used as positive control in the assay. DISCUSSION
The data presented in this study demonstrate the presence of two distinct types of SOD in B. abortus. The cyanide sensitivity of a portion of the total activity, as shown both by the enzymatic and native polyacrylamide gel assays indicates the presence of C u / Z n SOD. This is clearly confirmed by the epinephrine assay (Table 1 ). A significant portion of the SOD activity, which was estimated to be 20-30% by the epinephrine assay, appears to be insensitive to the action of cyanide and azide. As Fe SOD is very sensitive to azide, the second band that migrated similar to the E. coli Fe SOD may be Mn SOD. The presence of more than one SOD in bacteria is common, but the presence of C u / Z n SOD is rare. Only a few bacteria have been shown to have C u / Z n SOD (Bannister and Parker, 1985; Steinman, 1985; Steinman, 1987). Recently, the C u / Z n SOD gene from a strain ofB. abortus has been found to be 27% homologous to h u m a n and bovine C u / Z n SOD (Beck et al., 1990). Erythritol was also used as a carbon source because B. abortus, with the exception of strain 19, is known to have preferential ability to utilize erythritol even in the presence of higher concentrations of glucose in the m e d i u m (Keppie et al., 1969, Smith et al., 1962). This ability ofB. abortus strains had led to the speculation that the abortion in cattle is caused by the preferential replication of the bacterium in the gravid uterus, which is rich in erythritol (Smith et al., 1962). Our data showed that SOD activities were similar re-
N. SRIRANGANATHAN ET AL.
gardless of the carbon source and that SOD expression is not influenced by erythritol levels. Among the strains tested in this study, strain 2308 is the most virulent as defined by delayed clearance from mice (Montaraz and Winter, 1986) and cattle (Subcommittee on Brucellosis Research, 1977). Clearance of strain 19 is faster (Montaraz and Winter, 1986; Subcommittee on Brucellosis Research, 1977) and persists only for up to 6-7 weeks in mice (Ho and Cheers, 1982; Montaraz and Winter, 1986) and is an attenuated strain in cattle. Therefore, it is used as the official vaccine strain (Subcommittee on Brucellosis Research, 1977 ). Persistence of strain 45/20 is variable depending upon its unpredictable tendency to reverse to the smooth form (Ho and Cheers, 1982). Strain RB51 is of low relative virulence and is only able to persist in low numbers with minimal replication for a m a x i m u m of 4 weeks in mice inoculated with more than 108 organisms (Schurig et al., submitted). The SOD specific activity in crude extracts of Brucella strains is from 10 to 100 fold higher than that reported for other intracellular bacterial pathogens (Beaman et al., 1983; Mayer and Falkinham, 1986). However, the presence of large and similar amounts of SOD activities and lack of SOD activities in the culture supernatant among the B. abortus strains studied suggest that SOD is not a major virulence factor. This conclusion is based upon similar SOD activities in strain RB51 compared with those of other Brucella strains. Strain RB51 is essentially avirulent in mice and none of the strains tested excrete SODs into the culture supernatant. It may well be that SOD contributes to the virulence ofB. abortus but is only one of several factors (Canning et al., 1986). Others suggest that the presence of a novel C u / Z n SOD in Brucella may be a critical virulence associated factor which allows the survival of the organism in the host (Beck et al., 1990). At present, it is generally agreed that smooth strains which have the O-side chain of the LPS complex are most likely to be virulent B. abortus (Subcommittee on Brucellosis Research, 1977 ). Strain RB51 is devoid of the O-side chain and this deficiency may explain the low virulence regardless of the high content of SOD. Future studies which utilize SOD deficient mutants will clarify the relative importance of SODs as virulence factors in Brucella spp.
This material is based on work supported by grants from the U S D A / HATCH # 2126550 from Experimental Station at VPI & SU, USDA grant # 85-CRCR-l-1848 and USDA Competitive grant 88-37241-4131. The authors wish to acknowledge Kurt Neidigh, Delbert Jones, Kathy Reynolds and Tunu Misra for their technical assistance.
BRUCELLA S U P E R O X I D E D I S M U T A S E S
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