MolecularImmunology,Vol. 28, No. 112, pp. 35-39, 1991 Printedin Great Britain.

0161-5890/91 $3.00 + 0.00 PergamonPressplc

IMMUNOGLOBULIN G BINDING ACTIVITY BRUCELLA ABORTUS BETSY J. BRICKER,* LOUISA B. TABATABAI*

and

OF

JOHN E. MAYFIELD?

*U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA 50010, U.S.A. and tZoology Department, Iowa State University, Ames, IA 50011, U.S.A. (First received 29 February 1988; accepted in revisedform 12 February 1990) Abstract-A Erucella abortusprotein with a molecular weight of 50 kDa has been shown to bind bovine immunoglobulin G from healthy, brucellosis-free animals. The Brucella immunoglobulin G binding molecule appears to be a protein, since it is susceptible to proteolysis. The protein is presumed to be located on the cell surface, since intact cells precipitate bovine immunoglobulin G. Examination of other species of Brucellashows that all Brucelluspeciesand strains tested express the protein. B. abortuscells also bound immunoglobulin G from other animal species. These included cat, chicken, dog, guinea pig, horse, human, mouse, rat, sheep, swine, and turkey but not immunoglobulin G from goat or rabbit.

INTRODIJmION

Brucella abortus is a pathogen of cattle whose world-

wide incidence is on the rise (Alton and Plommet, 1986; Matyas and Fujikura, 1984). A serious problem for control and eradication programs is the high rate of false positive results in diagnostic tests (Chappel et al., 1978; Nicoletti, 1980). Current diagnostic assays involve screening bovine serum against a test antigen consisting of killed B. abortus cells (Alton et al., 1975; Moyer et nl., 1987; Nicoletti, 1980). Antibody binding to the test antigen is the indicator for exposure to the pathogen. The primary objective of the experiments reported here was to examine the incidence of non-immune binding of bovine and other animal immunoglobulin G (IgG) to a Brucella protein. Such binding activity is a potential source of interference in diagnostic procedures.

with an equal volume of sample buffer (2% SDS, 10% glycerol, 62.5% Tris and 0.001% bromophenol blue, pH 6.8), and boiled 5 min. The samples were then centrifuged (16,000g for 5 min; Eppendorf Micro Centrifuge Model 5415; Brinkman Inst., Westbury, NY) to remove the insoluble matter. Formalin-fixed Staphylococcus aureus (Cowan strain) was obtained from Sigma Chemical Company (St Louis, MO) as a 10% whole cell suspension. Cells were washed in saline prior to use. Antisera and IgG

Normal bovine sera were obtained from a nonvaccinated brucellosis-free herd (C. A. Belzer, MS Thesis, Iowa State University, Ames, IA, 1986). Biotin-labeled IgG (except turkey IgG), horseradish peroxidase (HRP)-labeled bovine Fc, HRP-labeled bovine Fab, and HRP-labeled streptavidin were obtained from Jackson Immunoresearch Labs (West Grove, PA) and reconstituted to 1 mg per ml. The HRP-conjugated goat antibody to bovine IgG (heavy chain-specific) was obtained from Kirkegaard and Perry Labs (Gaithersburg, MD) and reconstituted to 0.1 mg per ml. Biotin-labeled turkey IgG was prepared by precipitating IgG from turkey serum with 33% saturated ammonium sulfate, redissolving the pellet in H,O, and dialysing extensively against H,O followed by 0.1 M sodium bicarbonate (pH 8.3). Turkey IgG (1 mg) was conjugated with biotinamidocaproate Nhydroxysuccinimide (Sigma Chemical Company) by the method of Ogata (1988). The relative biotin labeling of each species’ IgG was titrated under standard assay conditions using HRP-streptavidin (see IgG precipitation assay below). All species of IgG except turkey had a similar titer with an apparent endpoint of detection at 3-6 ng

MATERIALS AND METHODS Cells

Brucella cell lines were cultured either on potato agar (B. abortus, B. canis, B. melitensis, B. neotomae and B. suis) or tryptose serum agar (B. abortus and B. ovis) at 37°C. Growth in 5% CO* was required for B. abortus strain 45120 and B. ovis,

infusion

while the other cells were grown in normal atmospheric conditions (Alton et al., 1975). Following a 48-72 hr growth period, the cells were harvested, washed in saline (0.85% NaCl), and standardized to a known cell density with a calorimeter (Bausch and Lomb, Inc., Rochester, NY) (absorbance 0.125 at 600 nm). Brucellu cells for precipitation experiments were heat killed at 65°C for 1 hr. Cells to be used for sodium dodecyl sulfate (SDSkpolyacrylamide gel electrophoresis were resuspended in saline, mixed 35

36

B. J.

BRICKER et al.

IgG per spot (1 mm x 8 mm slot). The endpoint of detection for turkey IgG was 12-25 ng IgG per spot. Antigens Brucellu hypertonic (Tabatabai

proteins (CSP) were extracted using a salt solution as previously described et al., 1979).

Western blot Total cell protein from Brucella cells denatured under non-reducing conditions was separated on 12.5% SDS acrylamide gels by the method of Laemmli (1970). Gels were pre-incubated in transfer buffer (12.4mM Tris, 96mM glycine, 0.05% SDS, and 20% methanol, pH 8.3) prior to electroelution of proteins onto nitrocellulose (NC) filter paper at 600mA for 1 hr (Transblot, Bio-Rad, Richmond, CA). After transfer, each NC filter was blocked with 3% fish gelatin (Norland Products, Inc., New Brunswick, NJ) in Tris buffered saline (TBS: 20 mM Tris, 500 mM NaCl, pH 7.5). The NC filters were incubated overnight at 4°C in bovine sera (diluted 1 to 50 in TBS containing 0.3% fish gelatin) from non-vaccinated brucellosis-free cows. After washing, each filter was incubated for 2 hr in HRP- conjugated goat anti-bovine IgG (diluted 1 to 500 in TBS containing 0.3% fish gelatin). After further washing, bound HRP-antibody was detected using a 0.06% 4-chloro-I-naphthol as substrate in a solution of TBS containing 0.012% hydrogen peroxide and 20% ethanol. Protease

treatment

Heat killed B. abortus (approximately 10” cells per sample) were suspended in 400 ~1 sample buffer without reducing agents, and boiled for 5 min. Eighty milligrammes of proteinase K (BoehringerMannheim Biochemistry, Indianapolis, IN) was added to one sample and incubated at 37°C for 5 hr. After pelleting the cellular debris, the supernatants were removed and reboiled for 5 min. Thirty microliters of each sample was loaded onto a 12.5% SDS-polyacrylamide gel and separated by electrophoresis. Western blotting was performed as described above. IgG precipitation

assay

Killed, intact bacterial cells (Escherichiu co/i, B. abortus, B. melitensis, all at 10 mg per ml wet weight; and S. aureus at 2.5 mg per ml wet weight) were washed three times in phosphate buffered saline (20 mm sodium phosphate, 150 mM sodium chloride, pH 7.4) prior to incubation in 1 ml of biotinconjugated IgG (100 pg per ml in phosphate buffered saline containing 0.3% fish gelatin) for 2 hr at room temp or overnight at 4C. The cells were washed free of the unbound IgG (three times in phosphate buffered saline), then resuspended in 50 ~1 (80 ~1 for B. melitensis) of diluted sample buffer (final concn of

0.5% SDS) for 5 min at room temp. Elution of IgG from the cells was necessary since Brucella cells do not adhere well to NC filter paper. The cells were removed by two centrifugation steps (16,OOOg for 5 min each). One microliter of each sample containing eluted IgG was spotted onto NC filter paper, blocked with 3% fish gelatin in TBS, and incubated with HRP streptavidin (1 pg per ml) at room temp for l-2 hr. The filters were washed and developed in 4-chloro-lnaphthol as described above. RESULTS

When 32 individual sera from non-vaccinated, brucellosis-free cows were reacted using the Western blot format with the total cellular protein of Bruce/la abortus, we found that bovine IgG consistently bound to a bacterial macromolecule approximately 50 kDa in size (data not shown). Binding of IgG to other B. abortus proteins was also observed in certain samples, but not as consistently or as strongly as the reaction with the 50 kDa molecule. To determine whether or not the B. abortus IgGbinding molecule was proteinaceous, the cells were lysed in sample buffer and digested with proteinase K. The binding of IgG is eliminated by protease treatment, indicating that the Brucellu binding molecule is either a protein or has an essential protein component. All six known species of Brucella were analyzed for expression of the binding protein. Whole cell lysates tested by the western blot technique show that the binding activity is expressed in all species (Fig. 1). Despite efforts to load constant amounts of bacterial protein in each lane, the different species and strains exhibited substantially different degrees of IgG binding by this technique. The protein was not found in the Brucella cell surface protein extract (CSP) (Fig. 1, lane B). Since Brucella species infect a variety of animals, the binding of IgG from other animals species to Brucella was examined. The amount of IgG from 14 animal species which bound to a constant amount of intact B. abortus strain 2308 cells is shown in Fig. 2. In this assay, the biotin-conjugated IgG bound to whole cells is eluted with an SDS solution and spotted onto NC and assayed by enzyme-linked immunosorbent assay. Since all cells were incubated with an equal concentration of biotin-conjugated IgG (100 pg per ml), the color intensity is related to the binding levels for each IgG. We observed a variety of binding levels for IgG ranging from negligible for goat and rabbit IgG to strong for horse, mouse, and sheep IgG. To compare the specificity of the B. melitensis binding activity with that of B. abortus, the assay was repeated using intact B. melitensis cells to bind goat and sheep IgG (Fig. 2, panel B). The results show that the B. melitensis and B. abortus have similar specificities for IgG from these two species.

IgG receptor

of 3. abortus

37

Fig. I. Expression of IgG binding activity in six Brucella species. The total cell protein from approximately IO9 cells of each Erucella cell line was screened by Western blot analysis using pooled normal bovine serum as primary antibody and HRP-labeled goat anti-bovine IgG as the detecting antibody. (A) B. abortus strain 2308 (smooth), (B) B. ahortus 3rucelia cell surface protein extract, (c) B. abortus strain S19 (smooth), (D) B. abortus strain 45/20 (rough), (E) 5. melitemis strain I6M, (F) B. suis strain Minn-X265, (G) B. oois strain NH-3572. (H) B. neotomae strain SE-I 169, (I) B. cams strain RM-6/66, and (J) B. abortus strain S19 (rough).

DISCUSSION

is located

A 50 kDa polypeptide which binds IgG from a variety of animal species has been identified in Bruceflu. It is inferred that the IgG binding activity

externally

on the bacterial

cell surface,

organisms

are capable

of precipitating

B S.aureusCONTROL lq6 CONTROL E.co/iCONTROL TURKEY SWME

since

IgG. If located on the surface, the protein must have a tight association with other surface components, since it is not detectable in the high salt Brucelfu cell surface intact

&mol.-GOAT &met.- SHEEP

SHEEP RAT RABBIT Fig. 2. Binding of IgG from various animals to whole cells. Each dot represents the total biotin-conjugated IgG eluted by 0.5% SDS from intact cells [200 pg B. abortw (strain 2308 rough); 200 @g E. eoli (strain 43591, 50 pg S. aureu~ (Cowan strain), or 125 pg B. me~itemis)], spotted onto NC filter paper. The NC bound biotin-IgG was detected with HRP-strepta~din. Panel A--B. abortus, panel B--B. melitensis. Three controis were included in panel A: E. coli control, precipitation of chicken IgG with E. co/i; IgG control, no IgG added; and S. aureus control, the precipitation of bovine IgG with S. aureus.

38

B. J. BRICKERet al.

protein extract (CSP) previously characterized by Tabatabai et al. (1979). This may account for the low background observed in the ELISA test based on CSP (Tabatabai and Deyoe, 1984). That the binding is of a non-immune nature is suggested by two lines of evidence: (i) IgG was bound from 32 individual cows previously designated as non-vaccinated brucellosis-free; and (ii) Brucella is able to precipitate IgG from a wide variety of animals including species not known to host natural infections (i.e. cat, chicken, guinea pig, mouse, rat and turkey). There is ample precedence for IgG binding proteins in other bacteria (reviewed by Boyle and Reis, 1987). The best known examples are Protein A from S. aureus and Protein G from the Group G streptococci. Most examples of IgG receptors involve grampositive bacteria (Forsgren and Sjoquist, 1966; Kronvall, 1973; Van de Merwe and Stegeman, 1985), although receptors on at least two gram-negative bacteria have been identified (Widders et al., 1985, 1988). It is not clear whether the IgG binding receptor of B. abortus is specific for the Fc portion of the antibody, as seen in other receptors, or if the Fab portion is involved. Although HRP-bovine IgG binds to the receptor, neither HRP-bovine IgG Fc nor HRP-bovine IgG Fab detectably bound (data not shown). Either the binding site is located very near the Fc-Fab cleavage site, or proteolytic cleavage affects the IgG structure thereby preventing binding. Although non-immune binding of bovine IgM and some IgG, has been previously described for Brucella (Nielsen and Duncan, 1982), no binding of bovine IgM by the 50 kDa protein was observed by western blot screening in this study. Furthermore, the 50 kDa binding activity reported here is not detectably affected by EDTA (data not shown) which was reported to destroy non-immune binding of bovine IgM and IgG, (Nielsen and Duncan, 1982). However, Nielsen and Duncan did not identify a Brucellu receptor responsible for this activity. The Brucellu IgG binding protein reported here was found to bind IgG from a variety of animal species, in agreement with the results reported for other IgG binding receptors. All previously reported receptors except that found on Taylorella have a high affinity for rabbit IgG, which is in direct contrast to the poor binding seen with Brucella binding protein. The Taylorella receptor binds only to horse and human IgG, and also differs from the Brucellu receptor. Two receptors have recently been described for Haemophilus somnus (Widders et al., 1988), which bind bovine antibody. The Haemophilus receptor which binds bovine IgG, is significantly larger (120-350 kDa) than the Brucella IgG binding protein, and the second receptor is slightly smaller (41 kDa). Thus, it appears that the Brucella receptor may be unique. Since the DNA or amino acid sequences for several IgG receptors are known, it will be interesting to compare these with the Brucellu

sequence for regions of homology or conservation of structure which might identify the binding site(s). Another unusual feature of the IgG receptor is that it appears to bind IgG from every animal which commonly hosts Brucella infections except for the goat. To address this further, since Brucella melitensis typically infects goats while Brucella abortus does not, we did the precipitation assay with B. melitensis cells to see if goat IgG would bind preferentially to the receptor. We thought that perhaps during the evolution of host preference, the receptor evolved an appropriate specificity. To the contrary, the same results were obtained when B. melitensis cells (originally isolated from a goat) were used (Fig. 2, panel B). In spite of host preference, B. melitensis has a low affinity for goat IgG, similar to the results observed for B. abortus (Fig. 2). The presence of the IgG binding protein on the B. melitensis cells was established by the binding of sheep IgG (Fig. 2, pane1 B). The IgG binding protein may be a virulence factor for B. abortus by allowing B. abortus to be coated with non-immune globulin. Furthermore, this protein may be a possible reason for false-positive results in agglutination-based serologic tests using whole Brucellu antigen. Development of a Brucellu test strain lacking this protein may eliminate the falsepositive reaction and, thus, increase the reliability of existing serologic tests. Acknowledgements-The authors would like to thank Dr B. L. Deyoe for kindly providing the BruceNa cultures. This investigation was supported, in part, by the Cooperative State Research Service under Agreement number 85XRCR-1-1842.

REFERENCES Alton G. G., Jones L. M. and Pietz D. E. (1975) Laboratory techniques in brucellosis, Second Edition Monograph, World Health Organization, Series No. 55, Geneva, Switzerland. Alton G. G. and Plommet M. (1986) Brucellosis summit in Geneva. WHO Chronicle 40, 19-21. Boyle M. D. P. and Reis K. J. (1987) Bacterial Fc receptors. Biotechnology 5, 697-703. Chappel R. J., McNaught D. J., Bourke J. A. and Allan G. S. (1978) Comparison of the results of some serologic tests for bovine brucellosis. J. Hyg. Camb. 80, 365-372. Forsgren A. and Sjoquist J. (1966) Protein A from S. aureus. I. Pseudo-immune reaction with human gamma globulin. J. Immun. 91, 822-827. Kronvall G. (1973) A surface component of group A, C, and G streptococci with non-immune reactivity for immunoglobulin G. J. Zmmun. 111, 1401-1406. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 68M85. Matyas Z. and Fujikura T. (1984) Brucellosis as a world problem. In Developments in Biological Standardization (edited by Valette L. and Hennesen W.), Vol. 56, pp. 3-20. S. Karger, Algiers, Algeria; Basil, Switzerland. Moyer N. P., Evins G. M., Pigott N. E., Hudson J. D., Farshy C. F., Feeley J. C. and Hausler W. J., Jr (1987) Comparison of serologic screening tests for brucellosis. J. clin. Microbial. 25, 1969-1972. Nicoletti P. (1980) The epidemiology of bovine brucellosis. Adv. Vet. Sci. Camp. Med. 24, 69-98.

IgG receptor of B. ubortus Nielsen K. and Duncan J. R. (1982) Demonstration that non-specific bovine Brucella ubortus agglutinin is EDTAlabile and not calcium-deoendent. J. Immun. 129. 366369. Ogata K. (1988) Immunovisualization with anti-antibodies: immunovisualization with peroxidase labeled antibodies. the peroxidase antiperoxihase, and biotin-streptavidin methods. In CRC Handbook of Immunoblotting of Profeins (edited by Bjerrum 0. J. and Heegaard N. H. H.). Vol. I, pp. 167-176. CRC Press, Boca Raton, FL. Tabatabai L. B. and Devoe B. L. (19841 Enzvme-linked immunosorbent assay ‘(ELISA) for the detection of bovine antibody to Brucella abortus with homologous salt-extractable protein antigens. J. clin. Microbial. 20, 209-2

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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, 668679.

Van de Merwe J. P. and Stegeman J. H. (1985) Binding of Coprococcus comes to the Fc portion of IgG. A possible role in the pathogenesis of Crohn’s disease. Eur. J. Immun. 15, 860-863. Widders P. R., Smith .I. W., Yarnall M., McGuire T. C. and Corbeil L. B. (1988) Non-immune immunoglobulin binding by Haemophilus somnus. J. Med. Microbial. 26, 307-3 11. Widders P. R., Stoke C. R., Newby T. .I. and Bourne F. J. (1985) Nonimmune binding of equine immunoglobulin by the causative organism of contagious equinine metritis, Taylorella equigenitalis. Infect. Immun. 48, 4 17-42 1.