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Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate — polyacrylamide gel electrophoresis
ELSEVIER
Veterinary Microbiology 39 (1994) 25-32
Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate- polyacrylamide gel electrophoresis Rodney A. Hill* and Donald R. Cook Rockhampton Veterinary Laboratories, Department of Primary Industries, Box 6014, Rockhampton Mail Centre, Queensland, Australia, 4702
(Accepted 8 June 1993)
Abstract The protein profiles of five field isolates of Brucella suis and four field isolates of B. abortus were examined. Isoelectric focusing of soluble proteins from cell sonicates produced 28 bands common to both species, with a further 10 bands being unique to a species. No intraspecies variation was detected. Sodium dodecyl sulfate extracted proteins were resolved by polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose and then stained. These techniques allowed the demonstration of 35 bands which were common to both species, with a further 15 bands which were unique to one of the two species. Again, no intraspecies variation was detected.
1. Introduction Brucella suis infection is endemic in feral pigs in certain areas in Queensland, Australia (Norton and Thomas, 1976). Where cattle and feral pigs live in close association, B. suis infection may be contracted by cattle, but cannot be differentiated from B. abortus infection in conventional serological tests ( C o o k and Noble, 1984). The presence of antibodies to B. suis complicates interpretation of results in the Australian bovine brucellosis eradication campaign, which can only be resolved by costly and time consuming investigation of the affected herd. The aim of this work was to determine whether the two species have unique proteins.
*Corresponding author, present address: Tropical Cattle Research Centre, CSIRO, Box 5545, RockhamptonMail Centre, Queensland, Australia. 4702. 0378-1135/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI0378- 1 135 (93) E0082-S
26
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
Such a unique protein, if immunogenic, would theoretically provide an ideal antigen for use in a species specific enzyme linked immunosorbent assay (ELISA). Isoelectric focusing (IEF) of B. abortus cell sonicates has been reported previously (Hatten and Brodeur, 1978, Tabatabai and Deyoe, 1984). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is a widely used technique for the isolation of brucellae cell envelope proteins (Dubray and Charriaut, 1983, Moriyon and Berman, 1982, Moriyon and Berman, 1983, Moriyon et al., 1987, Tabatabai et al., 1979, Verstreate and Winter, 1984, Winter, 1987). It appears that neither IEF nor SDS PAGE have been used to examine the proteins of B. suis.
2. Materials and methods Bacteria
Four smooth field isolates ofB. abortus biotype 1 and five smooth field isolates ofB. suis biotype 1 were used. Biotyping was done by the National Biological Standards Laboratory, Canberra, Australia. Cell suspensions
The isolates were grown on nutrient agar (Oxoid, United Kingdom, code CM3) supplemented with 1% ( w / v ) dextrose and 5% (v/v) sterile equine serum, for 48 h at 37°C in 9% CO2. Colonial morphology was checked by standard methods (Alton et al., 1975). Bacterial lawns were harvested by washing the cells off the plates with 0.02 M phosphate buffered saline (PBS) (pH 7.2). The cell suspensions were washed three times in PBS, then resuspended in PBS to give a final concentration of 0.1 g wet weight of cells/ml. The standardised whole cell suspensions (WCS) were then heat killed at 65°C for 60 min (Dubray and Bezard, 1980), and checked for sterility before being stored in 1 ml aliquots at - 20°C. All procedures involving live brucellae were performed in a Class II biohazard cabinet. Protein extraction
Killed cell suspensions were ultrasonically disrupted (Branson B30 Sonifier fitted with a step microtip) until 90% disruption, estimated by microscopic examination, was achieved. The cell suspensions were cooled in an icebath during sonication. Part of these sonicated suspensions (SS) were centrifuged to remove non-soluble material, and both the soluble fractions (SF) and the sonicated suspensions were stored at -20°C. Protein determinations
Protein levels in SF, WCS, and SS preparations were determined by the Lowry method (Lowry et al., 1951 ). For the WCS and SS preparations, the protein determination was performed after the preparations had been disolved in a modified Laemmli sample buffer (Laemmli, 1970) that lacked both 2-mercaptoethanol and bromophenol blue. Samples containing identical quantities of protein were applied to gels when protein bands were directly compared for qualitative and quantitative differences.
RodneyA. Hill/ VeterinaryMicrobiology 39 (1994)25-32
27
IEF
IEF was performed essentially as described in the Pharmacia Isoelectric Focusing Handbook (Anon, 1982). Briefly, 1% agarose gels (95 X 85 X 1 mm) containing 6.4% ampholytes, with a pI range 2.5-6.5 were prefocussed for 10 min. SF containing 80-300/xg protein for gel staining procedures and 8-24/xg protein for electroblotting, were applied on paper applicators and run for 90 rain at constant power of 4.0 watts. Samples were titrated over the stated ranges to determine the optimal protein level to apply to gels. The following pI standards were applied to each gel:- pepsinogen (pI 2.80), amyloglucosidase (pI 3.50), methyl red ( dye ) (pI 3.75 ), glucose oxidase ( pI 4.15 ), soybean trypsin inhibitor ( pI 4.55 ), fl-lactoglobulin A (pI 5.20), bovine carbonic anhydrase B (pI 5.85), and human carbonic anhydrase B (pI 6.55) (Pharmacia, Uppsala, Sweden). Gels prepared for staining procedures were fixed, stained for proteins with Coomassie blue and destained (Rosen and Lindstr6m, 1979). SDS PAGE
The discontinuous system (Laemmli, 1970) was used with horizontal slab gels ( 110 X 110 X 1.2 mm) containing 10% acrylamide in the separating gel. Both WCS and SS were prepared for SDS PAGE by mixing with Laemmli sample buffer to contain 80-200 ~g protein for gel staining procedures and 2-12/xg protein for electroblotting. Samples were titrated over the stated ranges to determine the optimal protein level to apply to gels. Reference proteins used were phosphorylase b (94000 molecular weight), bovine serum albumin (68000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100), and c~-lactalbumin (14400) (Pharmacia, Uppsala, Sweden). Gels were run at 40mA until the tracking dye was 2 mm from the anode. The Coomassie blue staining method (Fehrnstr6m and Moberg, 1977) was used. Electroblotting of SDS PAGE and IEF gels
Protein bands in SDS PAGE and IEF gels were transferred (LKB2005 Transphor Electroblotting Unit) to 0.45/z pore size nitrocellulose (NC) sheets essentially using the LKB method (Anon, 1983 ). They were stained using a biotin-avidin protein detection stain (BioRad Laboratories, Richmond, Calif.), referred to as total protein stain (TPS).
3. Results lsoelectric Focusing of sonicated extracts ofB. suis and B.abortus Thirty-eight protein bands were identified in SFs of B. suis and B. abortus. Coomassie
blue staining of IEF gels, and TPS on NC sheets, showed identical results. There was no variation in band pattern within each species, for the four B. suis and five B. abortus strains tested. Each species profile was characteristic and reproducible. IEF protein bands were designated F I-F38; numbered sequentially from the anode end. The protein profiles of the two species are compared in Fig. 1. There are both quantitative and qualitative differences between B. suis and B. abortus protein bands, as judged by staining intensity. Of the 28 protein bands common to both species, 13 show quantitative
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
28
abc
d
e
g
f
+ Fig. 1. Comparison of the protein bands separated from sonicated extracts (SFs) of Brucella suis (Lane A) and B. abortus (Lane B) by isoelectric focusing (pI range 2.5 to 6.5), stained with Coomassie blue. The pI markers were: a, pepsinogen (pI 2.80); b, amyloglucosidase (pI 3.50); c, methyl red (dye) (pI 3.75); d, glucose oxidase (pI 4.15) ; e, soybean trypsin inhibitor (pI 4.55) ; f,/3-1actoglobulin A (pI 5.20); and g, bovine carbonic anhydrase B (pI 5.85), (Pharmacia, Uppsala, Sweden). Table 1 Comparison of the pI values of the major protein bands separated from sonicated extracts of Brucella melitensis biovar Suis and B. melitensis biovar Abortus by isoelectric focusing (pI range 2.5 to 6.5), stained with Coomassie blue Protein Ident. pI
Fa I
F F 2 3 2.80
F 4
F 5
F 6
Suis
Lc L
M
L
L
L
-
M
L
H
M
L
L
L
L
L
L
-
Abortus
Protein Ident. pI
F F 20 21 4.87
Suis H Abortus H
F F 7 8 3.98
F F 9 10 4.05
F F 11 12 4.30
F F 13 14 4.47
H
L
M
L
M
L
L
M
L
H
L
M
F 32
F F 33 34 5.43
F 22
F F 23 24 4.95
F 25
F 26 5.08
F F 27 28 5.10
F 29
F F 30 31 5.28
L
L
M
-
L
L
L
L
-
M
M
L
L
L
-
M
L
L
M
M
L
L
L
M
L
M
F F 15 16 4.55
F F 17 18 4.72
F 19
M
M
L
-
L
L
L
M
F 35
F 36
F 37
Fb 38 5.67
L
M
L
M
L
M
~Band closest to anode. bBand closest to cathode. CDegree of staining with coomassie blue - L = Light, M = Medium, H = Heavy, - = No band detected.
variation. Ten protein bands are qualitatively different. The pI values of some of the major p r o t e i n b a n d s f r o m S F s o f B. suis and B. abortus are s h o w n in T a b l e 1.
SDS P A G E Electroblots o f SDS extracted proteins We found that TPS on NC sheets was more sensitive than Coomassie blue staining of S D S P A G E g e l s . S o m e p r o t e i n b a n d s p r e s e n t at l o w c o n c e n t r a t i o n s w e r e n o t d e t e c t e d i n Coomassie blue stained gels, but were detected with TPS. Therefore, Coomassie blue stained g e l s w e r e n o t s t u d i e d in d e t a i l . T h e S D S P A G E p r o t e i n b a n d p r o f i l e ( d e t e c t e d b y T P S o f
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
29
electroblots) of each Brucella species was characteristic and reproducible. There was no variation in band pattern within each species and no differences between SS and WCS. Fifty protein bands were detected using TPS. These were designated El-E50; numbered sequentially from the lowest molecular weight ( = 14 kDa) to the highest ( -~ 98 kDa). Forty protein bands were detected from B. suis extracts; and 45 bands from B. abortus extracts. Thirty-five bands were common to both species, with 11 showing quantitative differences
A
B
941(-" 671(4 3K-" 301(-" 20.11(1 4.4K-Fig. 2. Comparison of the SDS PAGE profiles of Brucella suis ( Lane A) and B. abortus ( Lane B ) ( 12/xg protein / lane), transblotted onto nitrocellulose sheet, detected with Biotin-avidin protein detection kit (Bio-rad, USA).
A
B
9 4K-"__ 6 7 K-" 4 3 K-" - -
30K~ 20.1K-" - -
14.41(-
Fig.3. Diagramatic representation of the major protein bands in SDS PAGE profiles of Brucella suis (Lane A) and B. abortus (Lane B), when transblotted onto nitrocellulose sheet, detected with Biotin-avidin protein detection kit ( Bio-rad, USA).
30
RodneyA. Hill/ VeterinaryMicrobiology 39 (1994)25-32
as judged by staining intensity. Five bands were unique to B. suis and 10 to B. abortus (Fig. 2). The major protein bands from SS and WCS orB. suis and B. abortus are shown in Fig. 3.
4. Discussion IEF of brucellae soluble fractions (SF) We found no reports of IEF applied to B. suis. Our IEF results for SF of B. abortus field isolates agree with those of Tabatabai and Deyoe (1984), who used B. abortus 2308 cell sonicates. Hatten and Brodeur (1978) conducted a similar study; in which they found a similar number of protein bands - 31 in B. abortus 23448 (biotype 1). However, the majority of components in their study were displaced towards the cathode in comparison to our results and those of Tabatabai and Deyoe (1984). They also investigated virulent strains of B. abortus biotypes 5 and 7; and attenuated strain 19; and found qualitative and quantitative differences amongst them. Fourteen bands of a total of 31 were common to all four strains. Our results show 28 bands of a total of 38; common to B. suis and B. abortus; and illustrates the degree of genetic homogeneity between these two species. SDS PAGE of brucellae whole cell suspensions (WCS) and sonicated suspensions ( SS). We found no reports of SDS PAGE of B. suis. Indistinguishable SDS PAGE profiles of SS and WCS indicate that the cells in the WCS were ruptured during preparation for electrophoresis. In other reports of SDS extracted brucellae proteins, different protocols were used (Dubray and Charriaut, 1983, Moriyon and Berman, 1983). Dubray and Charriaut found forty discrete bands in their SDS soluble cell wall fraction, with a major band at 43 kDa; their SDS insoluble fraction showing major bands at 25 kDa and 36 kDa. Moriyon and Berman (1983) found that SDS-/3Me at 100°C extracted a major protein at 38 kDa and smaller amounts of several other proteins. The variability between each of these and our protocols may partially explain the variability of results obtained. Minor variations in protein profiles of 23 isolates of B. abortus Biotype 1 were detected by Verstreate and Winter (1984). They also found minor variations between Biotypes 1, 2 and 4 of B. abortus. They identified three major clusters of outer membrane protein at 8894 k, 34-40 k and 25-30 k present in all 49 strains tested. Several other authors, using sequential extraction of physically disrupted brucellae with N-lauroylsarcosinate and dipolar ionic detergents (Douglas et al., 1984, Moriyon and Berman, 1982, Verstreate et al., 1982) have also isolated proteins with molecular weights similar to our major proteins; specifically, 25-30 k, 35-40 k, and 88-94 k (Douglas et al., 1984, Verstreate et al., 1982). Moriyon and Berman (1982) using B. abortus Strains 45/20 and 1119-3 found a range of proteins from = 10 k-94 k showing many heavy bands. They found that "Sarkosyl", Triton X100, and "Zwittergent 316" detergents were less effective than SDS for extraction of cell envelope proteins. Amongst those extracted by SDS were several proteins below 29 kDa and others at 30, 38, 40, 68, and 88 kDa, showing close agreement with our results for B. abortus. These different protocols reveal very similar major protein bands of B. abortus using SDS-PAGE. The single-step approach, using Laemmli buffer to extract brucellae proteins from WCS
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
31
and SS was effective. It p r o v i d e d reproducible extraction within each species o f Brucella. All m a j o r protein bands p r e v i o u s l y demonstrated in B. abortus w e r e present in this study. S o m e o f the m a j o r proteins o f B. suis and B. abortus appear to be different. H o w e v e r , in each species, S D S - P A G E blots reveal corresponding bands present in l o w e r quantities. This applies to m a j o r proteins o f B. abortus at 94, 6 8 - 7 0 , 48, 39, 32, and 20 kDa; and to B. suis at 47, 31, and 24 kDa. The less abundant proteins 15 of which appear to be unique to a species are present throughout the profiles with a m a j o r cluster b e t w e e n 43 and 67 kDa. The significance of these differences to the b o v i n e i m m u n e system, is a topic for further research. D e v e l o p m e n t o f a species specific E L I S A requires that the b o v i n e i m m u n e system must r e c o g n i s e these proteins as unique i m m u n o g e n s , if they are to be useful.
Acknowledgement This w o r k was supported by the Australian National Brucellosis and Tuberculosis Eradication Campaign.
References Alton, G.G., Jones, L.M. and Pietz, D.E., 1975. Laboratory techniques in brucellosis (2nd edition ), World Health Organization, Geneva. Anon, 1982. Isoelectric Focusing principles and methods, Pharmacia Fine Chemicals, Ljungfortagen. Anon, 1983. Laboratory Manual for LKB2005 Transfor Electroblotting Unit, LKB, Bromma. Cook, D.R. and Noble, J.W., 1984. Isolation ofBrucella suis from cattle. Aust. Vet. J., 61: 263-264. Douglas, J.T., Rosenberg, E.Y., Nikaido, H., Verstreate, D.R. and Winter, A.J., 1984. Porins of Brucella species. Infect. Immun., 44: 16-21. Dubray, G. and Bezard, G., 1980. Isolation of three Brucella abortus cell wall antigens protective in murine experimental brucellosis. Ann. Rech. Vet., 11: 367-373. Dubray, G. and Charriaut, C., 1983. Evidence of three major polypeptide species and tWO major polysaccharide species in the Brucella outer membrane. Ann. Rech. Vet., 14:311-318. Fehrnstr6m, H. and Moberg, M., 1977. Application note 306 - SDS and conventional polyacrylamide gel electrophoresis with LKB 2117 Multiphor, LKB, Bromma. Hatten, B.A. and Brodeur, R.A., 1978. Soluble antigens of virulent and attenuated biotypes of Brucella abortus. Infect. Immun., 22: 956-962. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the, head of the bacteriophage T4. Nature 227: 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with Folin-Phenol reagent. J. Biol. Chem., 193: 265-275. Moriyon, I. and Berman, D.T., 1982. Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J. Bact., 152: 822-828. Moriyon, I. and Berman, D.T., 1983. Isolation purification and partial characterization of Brucella abortus matrix protein. Infect. Immun., 39: 394-402. Moriyon, I., Gamazo, C. and Diaz, R., 1987. Properties of the outer membrane of Brucella. Ann. Inst. Pasteur/ Microbiol., 138: 89-91. Norton, J.H. and Thomas, A.D., 1976. Brucella suis in feral pigs. Aust. Vet. J., 52: 293-294. Rosen, A. and Lindstr6m, M., 1979. Application note 317 - Agarose isoelectric focusing of monoclonal IgM and IgG antibodies, LKB, Bromma. Tabatabai, L.B. and Deyoe, B.L., 1984. Characterization of salt extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectric focusing. Vet. Microbiol., 9: 549-560.
32
Rodney A. Hill/Veterinary Microbiology 39 (1994) 25-32
Tabatabai, L.B., Deyoe, B.L. and Ritchie, A.E., 1979. Isolation and characterization of toxic fractions from Brucella abortus. Infect. Immun., 26: 668-679. Verstreate, D.R., Creasy, M.T., Caveney, N.T., Baldwin, C.L., Blab, M.S. and Winter, A.J., 1982. Outer membrane proteins of Brucella abortus isolation and characterization. Infect. Immun., 35: 979-989. Verstreate, D.R. and Winter, A.J., 1984. Comparison of sodium dodecyl sulphate-polyacylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infect. Immun., 46: 182-187. Winter, A.J., 1987. Outer membrane proteins of Brucella. Ann. Inst. Pasteur/Microbiol., 138: 87-89.
Veterinary Microbiology 39 (1994) 25-32
Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate- polyacrylamide gel electrophoresis Rodney A. Hill* and Donald R. Cook Rockhampton Veterinary Laboratories, Department of Primary Industries, Box 6014, Rockhampton Mail Centre, Queensland, Australia, 4702
(Accepted 8 June 1993)
Abstract The protein profiles of five field isolates of Brucella suis and four field isolates of B. abortus were examined. Isoelectric focusing of soluble proteins from cell sonicates produced 28 bands common to both species, with a further 10 bands being unique to a species. No intraspecies variation was detected. Sodium dodecyl sulfate extracted proteins were resolved by polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose and then stained. These techniques allowed the demonstration of 35 bands which were common to both species, with a further 15 bands which were unique to one of the two species. Again, no intraspecies variation was detected.
1. Introduction Brucella suis infection is endemic in feral pigs in certain areas in Queensland, Australia (Norton and Thomas, 1976). Where cattle and feral pigs live in close association, B. suis infection may be contracted by cattle, but cannot be differentiated from B. abortus infection in conventional serological tests ( C o o k and Noble, 1984). The presence of antibodies to B. suis complicates interpretation of results in the Australian bovine brucellosis eradication campaign, which can only be resolved by costly and time consuming investigation of the affected herd. The aim of this work was to determine whether the two species have unique proteins.
*Corresponding author, present address: Tropical Cattle Research Centre, CSIRO, Box 5545, RockhamptonMail Centre, Queensland, Australia. 4702. 0378-1135/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI0378- 1 135 (93) E0082-S
26
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
Such a unique protein, if immunogenic, would theoretically provide an ideal antigen for use in a species specific enzyme linked immunosorbent assay (ELISA). Isoelectric focusing (IEF) of B. abortus cell sonicates has been reported previously (Hatten and Brodeur, 1978, Tabatabai and Deyoe, 1984). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is a widely used technique for the isolation of brucellae cell envelope proteins (Dubray and Charriaut, 1983, Moriyon and Berman, 1982, Moriyon and Berman, 1983, Moriyon et al., 1987, Tabatabai et al., 1979, Verstreate and Winter, 1984, Winter, 1987). It appears that neither IEF nor SDS PAGE have been used to examine the proteins of B. suis.
2. Materials and methods Bacteria
Four smooth field isolates ofB. abortus biotype 1 and five smooth field isolates ofB. suis biotype 1 were used. Biotyping was done by the National Biological Standards Laboratory, Canberra, Australia. Cell suspensions
The isolates were grown on nutrient agar (Oxoid, United Kingdom, code CM3) supplemented with 1% ( w / v ) dextrose and 5% (v/v) sterile equine serum, for 48 h at 37°C in 9% CO2. Colonial morphology was checked by standard methods (Alton et al., 1975). Bacterial lawns were harvested by washing the cells off the plates with 0.02 M phosphate buffered saline (PBS) (pH 7.2). The cell suspensions were washed three times in PBS, then resuspended in PBS to give a final concentration of 0.1 g wet weight of cells/ml. The standardised whole cell suspensions (WCS) were then heat killed at 65°C for 60 min (Dubray and Bezard, 1980), and checked for sterility before being stored in 1 ml aliquots at - 20°C. All procedures involving live brucellae were performed in a Class II biohazard cabinet. Protein extraction
Killed cell suspensions were ultrasonically disrupted (Branson B30 Sonifier fitted with a step microtip) until 90% disruption, estimated by microscopic examination, was achieved. The cell suspensions were cooled in an icebath during sonication. Part of these sonicated suspensions (SS) were centrifuged to remove non-soluble material, and both the soluble fractions (SF) and the sonicated suspensions were stored at -20°C. Protein determinations
Protein levels in SF, WCS, and SS preparations were determined by the Lowry method (Lowry et al., 1951 ). For the WCS and SS preparations, the protein determination was performed after the preparations had been disolved in a modified Laemmli sample buffer (Laemmli, 1970) that lacked both 2-mercaptoethanol and bromophenol blue. Samples containing identical quantities of protein were applied to gels when protein bands were directly compared for qualitative and quantitative differences.
RodneyA. Hill/ VeterinaryMicrobiology 39 (1994)25-32
27
IEF
IEF was performed essentially as described in the Pharmacia Isoelectric Focusing Handbook (Anon, 1982). Briefly, 1% agarose gels (95 X 85 X 1 mm) containing 6.4% ampholytes, with a pI range 2.5-6.5 were prefocussed for 10 min. SF containing 80-300/xg protein for gel staining procedures and 8-24/xg protein for electroblotting, were applied on paper applicators and run for 90 rain at constant power of 4.0 watts. Samples were titrated over the stated ranges to determine the optimal protein level to apply to gels. The following pI standards were applied to each gel:- pepsinogen (pI 2.80), amyloglucosidase (pI 3.50), methyl red ( dye ) (pI 3.75 ), glucose oxidase ( pI 4.15 ), soybean trypsin inhibitor ( pI 4.55 ), fl-lactoglobulin A (pI 5.20), bovine carbonic anhydrase B (pI 5.85), and human carbonic anhydrase B (pI 6.55) (Pharmacia, Uppsala, Sweden). Gels prepared for staining procedures were fixed, stained for proteins with Coomassie blue and destained (Rosen and Lindstr6m, 1979). SDS PAGE
The discontinuous system (Laemmli, 1970) was used with horizontal slab gels ( 110 X 110 X 1.2 mm) containing 10% acrylamide in the separating gel. Both WCS and SS were prepared for SDS PAGE by mixing with Laemmli sample buffer to contain 80-200 ~g protein for gel staining procedures and 2-12/xg protein for electroblotting. Samples were titrated over the stated ranges to determine the optimal protein level to apply to gels. Reference proteins used were phosphorylase b (94000 molecular weight), bovine serum albumin (68000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100), and c~-lactalbumin (14400) (Pharmacia, Uppsala, Sweden). Gels were run at 40mA until the tracking dye was 2 mm from the anode. The Coomassie blue staining method (Fehrnstr6m and Moberg, 1977) was used. Electroblotting of SDS PAGE and IEF gels
Protein bands in SDS PAGE and IEF gels were transferred (LKB2005 Transphor Electroblotting Unit) to 0.45/z pore size nitrocellulose (NC) sheets essentially using the LKB method (Anon, 1983 ). They were stained using a biotin-avidin protein detection stain (BioRad Laboratories, Richmond, Calif.), referred to as total protein stain (TPS).
3. Results lsoelectric Focusing of sonicated extracts ofB. suis and B.abortus Thirty-eight protein bands were identified in SFs of B. suis and B. abortus. Coomassie
blue staining of IEF gels, and TPS on NC sheets, showed identical results. There was no variation in band pattern within each species, for the four B. suis and five B. abortus strains tested. Each species profile was characteristic and reproducible. IEF protein bands were designated F I-F38; numbered sequentially from the anode end. The protein profiles of the two species are compared in Fig. 1. There are both quantitative and qualitative differences between B. suis and B. abortus protein bands, as judged by staining intensity. Of the 28 protein bands common to both species, 13 show quantitative
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
28
abc
d
e
g
f
+ Fig. 1. Comparison of the protein bands separated from sonicated extracts (SFs) of Brucella suis (Lane A) and B. abortus (Lane B) by isoelectric focusing (pI range 2.5 to 6.5), stained with Coomassie blue. The pI markers were: a, pepsinogen (pI 2.80); b, amyloglucosidase (pI 3.50); c, methyl red (dye) (pI 3.75); d, glucose oxidase (pI 4.15) ; e, soybean trypsin inhibitor (pI 4.55) ; f,/3-1actoglobulin A (pI 5.20); and g, bovine carbonic anhydrase B (pI 5.85), (Pharmacia, Uppsala, Sweden). Table 1 Comparison of the pI values of the major protein bands separated from sonicated extracts of Brucella melitensis biovar Suis and B. melitensis biovar Abortus by isoelectric focusing (pI range 2.5 to 6.5), stained with Coomassie blue Protein Ident. pI
Fa I
F F 2 3 2.80
F 4
F 5
F 6
Suis
Lc L
M
L
L
L
-
M
L
H
M
L
L
L
L
L
L
-
Abortus
Protein Ident. pI
F F 20 21 4.87
Suis H Abortus H
F F 7 8 3.98
F F 9 10 4.05
F F 11 12 4.30
F F 13 14 4.47
H
L
M
L
M
L
L
M
L
H
L
M
F 32
F F 33 34 5.43
F 22
F F 23 24 4.95
F 25
F 26 5.08
F F 27 28 5.10
F 29
F F 30 31 5.28
L
L
M
-
L
L
L
L
-
M
M
L
L
L
-
M
L
L
M
M
L
L
L
M
L
M
F F 15 16 4.55
F F 17 18 4.72
F 19
M
M
L
-
L
L
L
M
F 35
F 36
F 37
Fb 38 5.67
L
M
L
M
L
M
~Band closest to anode. bBand closest to cathode. CDegree of staining with coomassie blue - L = Light, M = Medium, H = Heavy, - = No band detected.
variation. Ten protein bands are qualitatively different. The pI values of some of the major p r o t e i n b a n d s f r o m S F s o f B. suis and B. abortus are s h o w n in T a b l e 1.
SDS P A G E Electroblots o f SDS extracted proteins We found that TPS on NC sheets was more sensitive than Coomassie blue staining of S D S P A G E g e l s . S o m e p r o t e i n b a n d s p r e s e n t at l o w c o n c e n t r a t i o n s w e r e n o t d e t e c t e d i n Coomassie blue stained gels, but were detected with TPS. Therefore, Coomassie blue stained g e l s w e r e n o t s t u d i e d in d e t a i l . T h e S D S P A G E p r o t e i n b a n d p r o f i l e ( d e t e c t e d b y T P S o f
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
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electroblots) of each Brucella species was characteristic and reproducible. There was no variation in band pattern within each species and no differences between SS and WCS. Fifty protein bands were detected using TPS. These were designated El-E50; numbered sequentially from the lowest molecular weight ( = 14 kDa) to the highest ( -~ 98 kDa). Forty protein bands were detected from B. suis extracts; and 45 bands from B. abortus extracts. Thirty-five bands were common to both species, with 11 showing quantitative differences
A
B
941(-" 671(4 3K-" 301(-" 20.11(1 4.4K-Fig. 2. Comparison of the SDS PAGE profiles of Brucella suis ( Lane A) and B. abortus ( Lane B ) ( 12/xg protein / lane), transblotted onto nitrocellulose sheet, detected with Biotin-avidin protein detection kit (Bio-rad, USA).
A
B
9 4K-"__ 6 7 K-" 4 3 K-" - -
30K~ 20.1K-" - -
14.41(-
Fig.3. Diagramatic representation of the major protein bands in SDS PAGE profiles of Brucella suis (Lane A) and B. abortus (Lane B), when transblotted onto nitrocellulose sheet, detected with Biotin-avidin protein detection kit ( Bio-rad, USA).
30
RodneyA. Hill/ VeterinaryMicrobiology 39 (1994)25-32
as judged by staining intensity. Five bands were unique to B. suis and 10 to B. abortus (Fig. 2). The major protein bands from SS and WCS orB. suis and B. abortus are shown in Fig. 3.
4. Discussion IEF of brucellae soluble fractions (SF) We found no reports of IEF applied to B. suis. Our IEF results for SF of B. abortus field isolates agree with those of Tabatabai and Deyoe (1984), who used B. abortus 2308 cell sonicates. Hatten and Brodeur (1978) conducted a similar study; in which they found a similar number of protein bands - 31 in B. abortus 23448 (biotype 1). However, the majority of components in their study were displaced towards the cathode in comparison to our results and those of Tabatabai and Deyoe (1984). They also investigated virulent strains of B. abortus biotypes 5 and 7; and attenuated strain 19; and found qualitative and quantitative differences amongst them. Fourteen bands of a total of 31 were common to all four strains. Our results show 28 bands of a total of 38; common to B. suis and B. abortus; and illustrates the degree of genetic homogeneity between these two species. SDS PAGE of brucellae whole cell suspensions (WCS) and sonicated suspensions ( SS). We found no reports of SDS PAGE of B. suis. Indistinguishable SDS PAGE profiles of SS and WCS indicate that the cells in the WCS were ruptured during preparation for electrophoresis. In other reports of SDS extracted brucellae proteins, different protocols were used (Dubray and Charriaut, 1983, Moriyon and Berman, 1983). Dubray and Charriaut found forty discrete bands in their SDS soluble cell wall fraction, with a major band at 43 kDa; their SDS insoluble fraction showing major bands at 25 kDa and 36 kDa. Moriyon and Berman (1983) found that SDS-/3Me at 100°C extracted a major protein at 38 kDa and smaller amounts of several other proteins. The variability between each of these and our protocols may partially explain the variability of results obtained. Minor variations in protein profiles of 23 isolates of B. abortus Biotype 1 were detected by Verstreate and Winter (1984). They also found minor variations between Biotypes 1, 2 and 4 of B. abortus. They identified three major clusters of outer membrane protein at 8894 k, 34-40 k and 25-30 k present in all 49 strains tested. Several other authors, using sequential extraction of physically disrupted brucellae with N-lauroylsarcosinate and dipolar ionic detergents (Douglas et al., 1984, Moriyon and Berman, 1982, Verstreate et al., 1982) have also isolated proteins with molecular weights similar to our major proteins; specifically, 25-30 k, 35-40 k, and 88-94 k (Douglas et al., 1984, Verstreate et al., 1982). Moriyon and Berman (1982) using B. abortus Strains 45/20 and 1119-3 found a range of proteins from = 10 k-94 k showing many heavy bands. They found that "Sarkosyl", Triton X100, and "Zwittergent 316" detergents were less effective than SDS for extraction of cell envelope proteins. Amongst those extracted by SDS were several proteins below 29 kDa and others at 30, 38, 40, 68, and 88 kDa, showing close agreement with our results for B. abortus. These different protocols reveal very similar major protein bands of B. abortus using SDS-PAGE. The single-step approach, using Laemmli buffer to extract brucellae proteins from WCS
Rodney A. Hill / Veterinary Microbiology 39 (1994) 25-32
31
and SS was effective. It p r o v i d e d reproducible extraction within each species o f Brucella. All m a j o r protein bands p r e v i o u s l y demonstrated in B. abortus w e r e present in this study. S o m e o f the m a j o r proteins o f B. suis and B. abortus appear to be different. H o w e v e r , in each species, S D S - P A G E blots reveal corresponding bands present in l o w e r quantities. This applies to m a j o r proteins o f B. abortus at 94, 6 8 - 7 0 , 48, 39, 32, and 20 kDa; and to B. suis at 47, 31, and 24 kDa. The less abundant proteins 15 of which appear to be unique to a species are present throughout the profiles with a m a j o r cluster b e t w e e n 43 and 67 kDa. The significance of these differences to the b o v i n e i m m u n e system, is a topic for further research. D e v e l o p m e n t o f a species specific E L I S A requires that the b o v i n e i m m u n e system must r e c o g n i s e these proteins as unique i m m u n o g e n s , if they are to be useful.
Acknowledgement This w o r k was supported by the Australian National Brucellosis and Tuberculosis Eradication Campaign.
References Alton, G.G., Jones, L.M. and Pietz, D.E., 1975. Laboratory techniques in brucellosis (2nd edition ), World Health Organization, Geneva. Anon, 1982. Isoelectric Focusing principles and methods, Pharmacia Fine Chemicals, Ljungfortagen. Anon, 1983. Laboratory Manual for LKB2005 Transfor Electroblotting Unit, LKB, Bromma. Cook, D.R. and Noble, J.W., 1984. Isolation ofBrucella suis from cattle. Aust. Vet. J., 61: 263-264. Douglas, J.T., Rosenberg, E.Y., Nikaido, H., Verstreate, D.R. and Winter, A.J., 1984. Porins of Brucella species. Infect. Immun., 44: 16-21. Dubray, G. and Bezard, G., 1980. Isolation of three Brucella abortus cell wall antigens protective in murine experimental brucellosis. Ann. Rech. Vet., 11: 367-373. Dubray, G. and Charriaut, C., 1983. Evidence of three major polypeptide species and tWO major polysaccharide species in the Brucella outer membrane. Ann. Rech. Vet., 14:311-318. Fehrnstr6m, H. and Moberg, M., 1977. Application note 306 - SDS and conventional polyacrylamide gel electrophoresis with LKB 2117 Multiphor, LKB, Bromma. Hatten, B.A. and Brodeur, R.A., 1978. Soluble antigens of virulent and attenuated biotypes of Brucella abortus. Infect. Immun., 22: 956-962. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the, head of the bacteriophage T4. Nature 227: 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with Folin-Phenol reagent. J. Biol. Chem., 193: 265-275. Moriyon, I. and Berman, D.T., 1982. Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J. Bact., 152: 822-828. Moriyon, I. and Berman, D.T., 1983. Isolation purification and partial characterization of Brucella abortus matrix protein. Infect. Immun., 39: 394-402. Moriyon, I., Gamazo, C. and Diaz, R., 1987. Properties of the outer membrane of Brucella. Ann. Inst. Pasteur/ Microbiol., 138: 89-91. Norton, J.H. and Thomas, A.D., 1976. Brucella suis in feral pigs. Aust. Vet. J., 52: 293-294. Rosen, A. and Lindstr6m, M., 1979. Application note 317 - Agarose isoelectric focusing of monoclonal IgM and IgG antibodies, LKB, Bromma. Tabatabai, L.B. and Deyoe, B.L., 1984. Characterization of salt extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectric focusing. Vet. Microbiol., 9: 549-560.
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Tabatabai, L.B., Deyoe, B.L. and Ritchie, A.E., 1979. Isolation and characterization of toxic fractions from Brucella abortus. Infect. Immun., 26: 668-679. Verstreate, D.R., Creasy, M.T., Caveney, N.T., Baldwin, C.L., Blab, M.S. and Winter, A.J., 1982. Outer membrane proteins of Brucella abortus isolation and characterization. Infect. Immun., 35: 979-989. Verstreate, D.R. and Winter, A.J., 1984. Comparison of sodium dodecyl sulphate-polyacylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infect. Immun., 46: 182-187. Winter, A.J., 1987. Outer membrane proteins of Brucella. Ann. Inst. Pasteur/Microbiol., 138: 87-89.