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Vol. 35, No. 3INFECTION AND IMMUNITY, Mar. 1982, p. 979-9890019-9567/82/030979-11$02.00/0

Outer Membrane Proteins of Brucella abortus: Isolation andCharacterization

D. R. VERSTREATE,* M. T. CREASY, N. T. CAVENEY, C. L. BALDWIN, M. W. BLAB, AND A. J.WINTER

Department of Clinical Sciences, New York State College of Veterinary Medicine, Cornell University, Ithaca,New York 14853

Received 27 July 1981/Accepted 6 November 1981

Outer membrane proteins were derived from one rough and four smooth strainsof Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and dipolar ionic detergent. Extraction of outer membraneproteins was ineffective, however, without predigestion with lysozyme. Threegroups of proteins were present and could be separated in their native state bysequential anion-exchange chromatography and gel filtration. Membrane proteinscontained substantial quantities of tightly adherent lipopolysaccharide whichcould be reduced but not eliminated by extraction of cells with trichloroacetic acidbefore disruption. Group 2 proteins, apparently trimers in their native state, gaverise to 43,000- and 41,000-molecular-weight bands after complete denaturation insodium dodecyl sulfate. They were antigenically identical among all the strains,showed close resemblance in amino acid composition to each other and a generalsimilarity to OmpF of Escherichia coli, and are proposed to be the porins of B.abortus. Group 3 proteins occurred as 30,000-molecular-weight bands on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, although additional bandswere frequently observed in this region. In none of the strains did group 3 proteinsmanifest heat-modifiable characteristics. Proteins of different strains bore a highdegree of similarity to each other in amino acid composition, except in methio-nine, isoleucine, tyrosine, and histidine. Differences occurred consistently inamino acid composition between group 2 and 3 proteins, and some of thesecorrespond to differences between OmpF and OmpA. Group 2 and 3 proteinswere antigenically distinct from each other, but the principal group 3 antigenswere shared among all the strains. Despite the lack of heat modifiability, perhapsinfluenced by adherent lipopolysaccharide, group 3 proteins are proposed ascounterparts to OmpA. Most of the group 1 proteins, minor components, werephysically associated with those of group 3 unless in sodium dodecyl sulfate.Group 1 proteins produced a major band at 94,000 and exhibited heat modifiabil-ity. No evidence was found of a low-molecular-weight lipoprotein in the outermembrane of B. abortus, but this is not taken to exclude its occurrence.

The principal classes of structural proteins inthe outer membrane of Escherichia coli arematrix porins (OmpC and OmpF), a heat-modifi-able protein (OmpA), and murein lipoprotein (6,45). With certain modifications, as, for example,in the absence of murein-linked lipoprotein (22,36, 57), counterpart proteins have been detectedin a wide variety of gram-negative genera (4, 6,8, 15, 21, 25, 26, 28, 34, 37, 53) and appear to beessential constituents of gram-negative bacteria.Virtually no information is yet available on thenature of comparable proteins in Brucella abor-tus. The extraction by sequential treatment withN-lauroylsarcosinate and dipolar ionic detergent(Zwittergent) of a protein presumably analogousto porin was presented in a preliminary report (I.Moriyon and D. T. Berman, Abstr. Annu. Meet.

Am. Soc. Microbiol. 1981, K171, p. 166), andKreutzer and Robertson (30) reported the ex-traction from B. abortus of a murein-associatedlipoprotein, although characterization of thissubstance was incomplete.Our interest in proteins of the outer membrane

of B. abortus stems from problems associatedwith eradication of brucellosis in the UnitedStates. The development of an effective subcel-lular vaccine devoid of lipopolysaccharide (LPS)might resolve the single greatest obstacle toeradication: detection of latently infected ani-mals (43). Additionally, diagnostic tests not reli-ant on the measure of LPS antibody would be ahighly desirable alternative, although progresshas been made in distinguishing vaccinationfrom infection titers by decreasing the vaccine

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dose (3) and by the observation that most infect-ed cattle produce a response to the native haptenof LPS not detectable in vaccinated animals (16,39). Although the proteins under investigationare structural constituents of the cell, and there-fore unlikely to function primarily as virulencefactors, they may induce immune responsesuseful in diagnosis and in conferring protectiveimmunity. Outer membrane proteins have beenused in other genera in serological classification(7, 8, 18, 24) and as vaccines (1, 13, 31). BecauseB. abortus is a facultative intracellular parasitedevoid of capsule, flagella, and pili, it seemedreasonable that protective immunity would in-clude a cell-mediated immune response to one ormore protein constituents of the cell wall. Thepurpose of this study was to isolate and charac-terize outer membrane proteins of B. abortusand to compare proteins among strains used forvaccination and representative virulent field iso-lates.

MATERIALS AND METHODSBacterial strains and cultivation. Five strains of

biotype 1 were used: two were vaccine strains usedcommercially in the United States (strain 19) and inother countries (strain 45/20), and three were virulentfield strains of diverse geographic origin (Table 1).Strain 2308 was isolated in 1942 and has been used inexperimental infection trials for over 30 years (35).The culture of 2308 that was used had been passagedtwice each in cows and guinea pigs, but only four timesin total in vitro (B. L. Deyoe, personal communica-tion). Strains C-10 and Y originated from Ontario andNew York State, respectively, and had been passagedin vitro no more than two times. In our laboratory,each strain was streaked over the surface of Schaedleragar plates (BBL Microbiology Systems, Cockeys-ville, Md.) containing 5% bovine blood and incubatedfor 2 days at 37°C in an atmosphere of air containing10%O CO2. Growth from several plates was suspendedin Albimi broth (Difco Laboratories, Detroit, Mich.),pooled, and frozen in portions at -70°C. For eachstrain, the same batch of stocks was used throughoutthis study.For batch cultivation, stock cultures were trans-

ferred successively onto Schaedler agar plates andAlbimi agar slants. Two-day growth from slants was

suspended in Albimi broth. Flasks were incubated in ashaker incubator (New Brunswick Scientific Co., NewBrunswick, N.J.) at 175 rpm and continuously gassed

at a rate of 1.0 cubic foot (28.317 cubic decimeters) perhour with a mixture of 95% air-5% CO2. Growth was

terminated at 24 h (corresponding to late-logarithmicphase) by placing flasks in ice, and cells were collectedby centrifugation at 4°C and washed twice in sterilephosphate-buffered saline (PBS) (pH 7.2). Because ofthe hazard of human infection, organisms were killed,unless otherwise noted, in PBS containing 0.5% For-malin. Killed cells were washed twice in sterile PBS,and the drained pellet was held at -20°C until extract-ed. Purity of growth from batch harvests was verifiedby examination of plates heaily streaked from thepooled flask contents. Colonial morphology was deter-mined by standard methods (2).

Extraction and purification of outer membrane pro-teins from physically disrupted ceUls. Cells were sus-pended at 1 g (wet weight) per 20 ml of 10 mM Tris-hydrochloride buffer (pH 7.5), and 1 mg each ofDNaseand RNase (Sigma Chemical Co., St. Louis, Mo.) wasadded per 100 ml. Disruption was accomplished bytwo passages through a high-pressure cell (Sorvall Ribicell fractionator, model RF-1) at 40,000 lb/in2, with thetemperature at the needle valve orifice maintained at10 to 15°C. The disrupted suspension was centrifugedat 3,000 x g for 20 min at 4°C and the supernatant wascentrifuged at 150,000 x g (average) for 60 min at 4°Cto pellet the crude membranes, which were resus-pended at 10 to 20 mg of protein per ml in Tris buffer.In some cases, outer membranes were separated fromcytoplasmic membranes by density gradient centrifu-gation (49).

Detergent extraction of cytoplasmic membraneswas performed by using either Triton X-100 (SigmaChemical Co.) (50) or sodium N-lauroylsarcosinate(Pfaltz & Bauer, Stamford, Conn.) (25). The resultantinsoluble material was dialyzed against Tris buffer at4°C for 72 h with repeated changes.The outer membrane-rich fraction, unless otherwise

noted, was subjected to digestion overnight at 37°Cwith egg white lysozyme (Mann Laboratories, NewYork, N.Y.) (1 mg/50 mg of membrane protein).Solubilization was then performed by using Triton X-100 with EDTA (50) or sodium deoxycholate (25),except that the protein concentrations used were 1 mg/ml and the extraction was performed at 37°C for 1 h.Zwittergent 3-14 (Calbiochem, La Jolla, Calif.) (0.2%)in Tris buffer containing 0.25 M NaCl was used underthe same conditions for this purpose. After extraction,the samples were centrifuged at 100,000 x g (average)for 20 min at 4°C, and the supernatants were held at40C.

Solubilized membrane fractions were concentratedby lyophilization to 10 to 20 mg/ml, equilibrated with10 mM Tris buffer containing 0.1% Zwittergent and

TABLE 1. Derivation and properties of B. abortus strainsStrain Colony type Description Origin19 Smooth Vaccine strain Biologics Division, U.S. Dept. of Agriculture45/20 Rough Vaccine strain National Animal Disease Center, Ames, Iowa2308 Smooth Field strain; from aborted fetus National Animal Disease Center, Ames, IowaC-10 Smooth Field strain; from supra- Animal Disease Research Inst., Nepean,

mammary lymph node Ontario, CanadaY Smooth Field strain; from aborted fetus N.Y. State Diagnostic Laboratory, Ithaca,

N.Y.

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0.25 M NaCl and applied to a column of DEAE-Sephacel (Pharmacia Fine Chemicals, Inc., Pis-cataway, N.J.) equilibrated with the same buffer.Elution was performed at room temperature withupward flow at a rate of 2 ml cm-2 h-1. After theinitial wash, a gradient of NaCl (0.25 to 0.75 M) wasestablished and collected over a period of 24 h. Proteinsamples from the ion-exchange column, concentratedby lyophilization, were separated under the sameconditions of flow on a column of Sephacryl S-300(Pharmacia Fine Chemicals, Inc.) equilibrated with 10mM Tris buffer containing 0.1% Zwittergent and 0.25M NaCl. If the proteins were not pure as judged bysodium dodecyl sulfate (SDS)-polyacrylamide gel elec-trophoresis (PAGE), they were either rechromato-graphed on Sephacryl S-300 or chromatographed withupward flow at 4 ml cm-2 h-1 on a column of hydrox-ylapatite (Biogel HT; Bio-Rad Laboratories, Rich-mond, Calif.), using a gradient of disodium phosphate(0.01 to 0.5 M).

Extraction of outer membrane proteins with SDS.Living or Formalin-killed cells were extracted sequen-tially at 60 and 100°C with buffer containing 2% SDS(48). In one experiment, Formalin-killed cells weresubjected to exhaustive extraction (17 h, with freshextraction buffer at 1- or 2-h intervals) at 60°C in 4%SDS, until no further proteins were detectable onSDS-PAGE of a 10-fold-concentrated supernatant.The residue was subjected to lysozyme digestion andZwittergent extraction under conditions outlined pre-viously. In another experiment, living and Formalin-killed cells were boiled for 4 h in the presence of 4%SDS (at 1-h intervals, with fresh extraction buffer eachtime), after which no proteins were detectable inconcentrated supernatants, and a portion of the insolu-ble residue, after thorough washing, was digestedovernight at 37°C with lysozyme (1 mg/60 mg [dryweight]).

Extraction of LPS. Acetone-dried cells were extract-ed with 45% hot phenol (56), and aqueous extraction ofthe phenol-soluble phase was performed by the meth-od of Moreno et al. (38, 39) to obtain fraction 5, acrude preparation of biologically active LPS, whichcontained 43% protein, 24% carbohydrate, and 0.6%2-keto-3-deoxyoctulosonic acid (KDO). The 50o le-thal dose of this preparation in 16- to 18-g maleHA(ICR) mice was 1.5 mg.

Trichloroacetic acid (TCA) extractions of wholecells, or of cytoplasmic constituents derived from the150,000 x g supernatant after pressure cell disruption,were performed by the method of Diaz et al. (16) andhydrolysis of TCA extracts by the method of Morenoet al. (39).SDS-PAGE. An equal volume of double-strength

extraction buffer (48) was added to each sample, andheating, unless noted otherwise, was performed for 10min at 100°C. In some instances, 0.1 M MgCl2 wasincluded in the extraction buffer in an effort to visual-ize the low-molecular-weight lipoprotein (21). SDS-PAGE was performed by the method of Laemmli (32)on 10 or 12.5% acrylamide gels. Phosphorylase b(94,000 molecular weight), bovine serum albumin(68,000), ovalbumin (43,000), carbonic anhydrase(30,000), soybean trypsin inhibitor (21,000), and lyso-zyme (14,300) (Bio-Rad Laboratories) were used asreference proteins. Gels were stained with Coomassieblue R-250 (Sigma Chemical Co.), and molecular

weight calculations were made from densitometricscans of Polaroid negatives (M. T. Creasy, submittedfor publication).

Chemical analysis. Amino acid analyses were per-formed on a Beckman model 119 CL analyzerequipped for single-column analysis. Samples werehydrolyzed in constant boiling HCl (5.7 N) containing0.1% ethanethiol for 24 h at 110°C. Protein was deter-mined by Peterson's modification of the method ofLowry et al. (47), with bovine serum albumin (MilesLaboratories, Inc., Elkhart, Ind.) as the standard.Total carbohydrates were estimated by the phenolsul-furic acid method (17) with glucose as a standard. Theassay for 2-keto-3-deoxy sugars described by Weiss-bach and Hurwitz (55) was performed, using Osborn'smodification (44). Results of this assay were taken as ameasure of KDO in the sample, and KDO (SigmaChemical Co.) was used as a standard. Correction for2-deoxyaldoses was made by the method of Warren(54).

Antisera. Male Flemish giant-chinchilla crossbredrabbits of 6 to 8 lb (-2.7 to 3.6 kg) were immunizedwith 2 to 3 mg of purified outer membrane protein in 1ml of Tris buffer emulsified in an equal volume ofcomplete Freund adjuvant. Half of the emulsion wasinjected intradermally in multiple sites on the back; theother half was injected intramuscularly in equal partsinto each hind leg. Three weeks later, 2 mg of proteinin buffer was injected intravenously. Animals wereexsanguinated 1 week thereafter. Two animals wereimmunized with each preparation. LPS antibodieswere removed by absorption with glutaraldehyde-fixed whole cells of strain 19, followed usually by theaddition of TCA extract which contained LPS andnative hapten. Absorption was considered completewhen no lines developed with TCA extract in immuno-diffusion reactions after 2 days of incubation at 23°C.A pool of antiserum with an agglutination titer in

excess of 1:400 was obtained from four heifers 1 to 4months after subcutaneous vaccination with strain 19.Sera from 11 cows infected with virulent field strainswere obtained from J. R. Duncan (ADRI, Ontario,Canada).

Immunological techniques. Immunodiffusion and im-munoelectrophoresis were performed on gels com-posed of 1% agarose (Sea Plaque, Marine Colloids,Inc., Rockland, Maine) in 0.03 M barbital buffer (pH8.8). In most immunodiffusion tests with bovine sera,NaCl was added to a concentration of 10o (16).Immunodiffusion tests were done with the use oftemplates (40). Immunoelectrophoresis was performedat 10 mA per frame in a Gelman chamber (GelmanInstrument Co., Ann Arbor, Mich.). Gels were driedand stained with 0.25% Crocein scarlet (Bio-Rad Lab-oratories) and 0.014% Coomassie blue R (SigmaChemical Co.) (14). Detergent concentrations in anti-gen solutions were less than 0.05% to avoid artifactswhich occurred at concentrations greater than 0.1%.

Statistical methods. The t test (52) was used tocompare the moles percent of each amino acid residuebetween group 2 and 3 proteins. The Spearman rankcorrelation method (52) was used to measure thedegree of similarity between B. abortus and E. coliouter membrane proteins in amino acid composition.By this method, the rank correlation (re) can rangefrom -1, signifying complete discordance, to +1,indicating complete concordance.

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RESULTSExtraction with Zwittergent and purification of

outer membrane proteins. Density gradient cen-trifugation of crude membranes resulted in abanding pattern similar to that described bySchnaitman (49). The concentration of KDO toprotein increased almost fivefold in the lowerband relative to the starting material (from 0.80to 3.85 ,ug/mg), indicating an enrichment in outercell membranes. The sarcosinate-insoluble ma-terial from the lower band and from a portion ofthe same preparation not processed throughgradient centrifugation was digested with lyso-zyme and extracted with Zwittergent. Each ofthe extracts contained three principal clusters ofproteins on SDS-PAGE, of which the dominantbands had molecular weights of 94,000, 43,000,and 30,000 (Fig. 1). These will be referred to asgroups 1, 2, and 3, respectively. The band at14,000 was lysozyme, but no other low-molecu-lar-weight bands were resolved on 10 or 12.5%gels, even with MgCl2 in the extraction buffer.Thus, group 1, 2, and 3 proteins were associatedwith the outer cell membrane, but gradient cen-trifugation was not a necessary condition fortheir extraction. In subsequent studies, this stepwas omitted. It was also demonstrated consis-tently that sarcosinate extracted cytoplasmicmembrane constituents more completely thandid Triton X-100 but did not remove outermembrane proteins, as has been reported for E.coli (12). Sarcosinate was therefore used in allsubsequent extractions.Comparison of solubilizing efficiency on sar-

cosinate-insoluble outer membrane proteins ofTriton X-100 + EDTA, sodium deoxycholate,and Zwittergent 3-14, with and without lyso-

ab94 K--

43K30K-'

14K--

FIG. 1. SDS-PAGE of B. abortus strain 19 outermembrane proteins. (a) Crude membrane preparationsubjected to density gradient centrifugation, extrac-tion with sarcosinate, lysozyme digestion, and extrac-tion with Zwittergent. (b) Same treatments with omis-sion of gradient centrifugation. The band at 14,000(14K) is lysozyme.

TABLE 2. Solubilizing efficiencies of detergentswith or without lysozyme predigestion on strain 19

sarcosinate-insoluble outer membrane proteins

Protein solubilized (%)Detergent No lysozyme Lysozyme

predigestion predigestion

2% Triton X-100 + 5 mM 0.9 73.4EDTA

2% Sodium deoxycholate 5.6 26.60.2% Zwittergent 3-14 5.0 77.8

zyme predigestion, demonstrated that lysozymepretreatment greatly enhanced the solubilizingefficiency of the detergents (Table 2). Of equalimportance, it was noted that group 3 proteinswere virtually absent from extracts preparedwithout lysozyme predigestion (Fig. 2c). Zwit-tergent was chosen for subsequent studies be-cause of its solubilizing efficiency and conve-nience.Outer membrane proteins from the five strains

of B. abortus contained group 1, 2, and 3 pro-teins (Fig. 3), and ion-exchange chromatog-raphy was highly effective in their separation.Although there was a general pattemn of elution,variations occurred among strains and evenamong different extracts of the same strain.Such variations may have been owing in somemeasure to micelle-protein interactions. In allseparations, the bulk of group 2 proteins ofsmooth strains (Fig. 4a and b) eluted at a con-ductivity at least 0.4 mS higher than those from

a b d e

SeK.:-

FIG. 2. SDS-PAGE of B. abortus strain 19 extract-ed with SDS. (a) Supernatant after extraction at 600Cfor 30 min. (b) Sediment from (a) heated for 10 min at1000C with dominant band at 41,000 (41K). (c) Com-parison with outer envelope proteins solubilized bydeoxycholate without prior lysozyme digestion. Group2 bands at 43,000 and 41,000 are evident, with anextremely faint band at 30,000. (d) Cells exhaustivelyheated in SDS at 60°C, then digested with lysozymeand extracted with Zwittergent, yielding bands at94,000, 43,000, 41,000, and 30,000. (e) Cells exhaus-tively heated in SDS at 100°C, then digested withlysozyme; only the 41,000 band of the outer membraneproteins is resolved. The lowest band in (d) and (e) islysozyme.

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FIG. 3. Densitometric scans from Polaroid negatives of SDS-PAGE gels of Zwittergent-soluble outermembrane preparations of B. abortus strains 19 (a), 45/20 (b), 2308 (c), Y (d), and C-10 (e). In (b) and (e), 30,000and 27,000 bands are resolved in group 3. Absorption at 580 nm is recorded on the ordinate. Numbers refer toprotein groups.

rough strain 45/20 (Fig. 4c). Group 3 proteinswere eluted with similar frequency before (Fig.4b) or after (Fig. 4a) application of the gradientand sometimes in several discrete peaks (Fig.4c). A portion of group 1 and 3 proteins wasalways associated in Zwittergent and could notbe separated completely by sequential gel filtra-tion and hydroxylapatite chromatography. SDSapparently disrupted this association, even atroom temperature (Fig. 5a). In general, theprocess of extraction and purification of roughstrain 45/20 was the easiest (and most complete),that of strain 19 was intermediate in difficulty,and that of the virulent strains was the mostdifficult. Difficulties encountered with smoothstrains may have been owing in part to interac-tions of LPS, native hapten, or other cell wallconstituents with outer membrane proteins.KDO was demonstrable in each of selectedpreparations tested (Table 3) and 0 antigenswere detectable by immunodiffusion in all of thepurified protein preparations, although veryfaintly in those of strain 45/20. Physical associa-tion of these proteins with LPS or native haptenwas indicated by lack of demonstrable free LPSor native hapten on immunoelectrophoresis(Fig. 6a, b, c, and d) and by inability to separateLPS or native hapten from group 2 proteinsthrough sequential ion-exchange, gel filtration,and hydroxylapatite chromatography. Extrac-

TABLE 3. Chemical analyses of purified outermembrane proteins of B. abortus

Strain Protein group KDO (ig/mg of protein)

45/20 2 2.345/20 3 13.8Y 2 2.3Y 3 4.2

tion of cells with TCA (16) before physicaldisruption and detergent extraction was found toreduce substantially (Fig. 6c and d) but not toeliminate 0 antigens bound to the membraneproteins. Proteins so derived were used as anti-gens in the later portion of this study. In E. coli ithas been shown that LPS is critical for themacromolecular association of porins (20, 45,58), but it was not possible in this study todistinguish to what degree LPS association withouter membrane proteins occurred in the intactcell or was a consequence of the extractionprocess. More drastic efforts to separate 0 anti-gens from purified B. abortus proteins were notmade because we believed that meaningful stud-ies of antigenic relationships could be done onlywith proteins in their native configuration.

Extraction of cells with SDS. Heating at 60°Creleased many proteins from the cell (Fig. 2a),including in some instances a faint band at43,000. When the sediment after a 60°C heatingwas heated in SDS at 100°C for 10 min (48), the41,000 band was the only one prominently visu-alized (Fig. 2b). Lysozyme digestion and Zwit-tergent extraction of the sediment after heatingfor 17 h at 60°C in 4% SDS caused release of allthe principal outer envelope proteins, includingbands at 94,000, 43,000, 41,000, and 30,000 (Fig.2d). Without lysozyme digestion, even 4 h ofheating at 100°C in 4% SDS failed to remove allof the outer membrane proteins from the mu-rein. Group 3 proteins were neither detected inconcentrated supernatants of 4-h boiled cells norresolved on SDS-PAGE of the sediment (Fig.2e). The amino acid composition of this sedi-ment resembled a mixture of group 2 and 3proteins. The same results were obtained withSDS extraction procedures whether performedwith living or with Formalin-killed cells.Amino acid analyses. Amino acid composi-

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FRACTION NUMBER

FIG. 4. Ion-exchange chromatography on DEAE-Sephacryl of Zwittergent-soluble outer membranepreparations of B. abortus strains 19 (a), Y (b), and 45/20 (c). Numbers refer to dominant protein groups ineach peak; L refers to lysozyme.

tions of group 2 proteins of the five B. abortusstrains were very similar. The same held true forgroup 3 proteins, except for large differences formethionine, isoleucine, tyrosine, and histidine(Table 4). The moles percent of tyrosine, phenyl-alanine, threonine, serine, glycine, and valinewere significantly greater, and those of proline,leucine, and lysine were significantly lower (P <0.05) in group 2 compared with group 3 proteins.It is notable that tyrosine and phenylalanine areproportionally more abundant and proline is less

abundant in OmpF than in OmpA of E. coli(Table 4). Rank correlations showed that al-though all of the B. abortus and E. coli outermembrane proteins were significantly similar (P< 0.01), a higher degree of similarity existedbetween B. abortus group 2 and E. coli OmpF (r,= 0.959) and between B. abortus group 3 and E.coli OmpA (rs = 0.918). The hydrophobicities (5)of group 2 and 3 proteins were 892 ± 30 caloriesper mol (3,732.5 ± 126 J/mol) and 980 ± 54calories per mol (4,101.1 ± 226 J/mol), respec-tively, and a polarity index (9) of 42% wascalculated for both protein groups, approximat-ing values for outer membrane proteins of other

; gram-negative bacteria (27, 48).E Heat modifiability. Purified group 2 and 3E proteins from each strain and partially purifiedE

group 1 proteins from strain 45/20 were incubat-H ed in 2% SDS for 2 h at 23 and 37°C and for 10> min at 100°C to determine effects on migration inF SDS-PAGE. Group 2 proteins were at approxi-D mately 115,000 after treatment at 23 or 37°C anda at 43,000 after heating at 100°C (Fig. Sd and e).zO After heating at 100°C, distinct bands were al-

ways present at 43,000 and 41,000 (Fig. 1 and 2c)unless gels were overloaded.Group 3 proteins displayed no heat modifiabil-

ity. A band was always present at 30,000 (Fig. 1and 2d) and in some preparations another ofvarying intensity at 27,000 (Fig. 4 and 5f and g).Additional minor bands sometimes observed inthis region (Fig. 1 and 5) varied in occurrenceamong extracts of the same strain.At 23°C, group 1 protein bands ranged from

approximately 84,000 to 97,000, with the heavi-est band at 84,000. After being heated at 37°C,the major band was at 94,000. Heating at 100°C

ea b c dK * .4. .. . >e

11 5K L3... i_'.:.:. & i94K-_"W., ,

43K--- i... .. -

t 9s: ::.t ...

xE, --

t .....

..

mm ::;-

.

FIG. 5. SDS-PAGE of group 1 (lanes a, b, and c),group 2 (lanes d and e) and group 3 (lanes f and g)proteins of B. abortus strain 45/20 after differenttemperature treatments. Proteins were incubated in2% SDS for 2 h at 23'C (lane a), for 2 h at 37'C (lanes b,d, and f), and for 10 min at 100°C (lanes c, e, and g).Group 1 and 3 preparations contain small quantities ofthe opposite protein species.

EC

0cli0

z

m0(c::(/)m0t

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OUTER MEMBRANE PROTEINS OF B. ABORTUS 985

caused further intensification of the 94,000bands, disappearance of bands above 94,000 andgeneration of several lower bands (Fig. 5a, b,and c). The faint bands in each strain presentbetween the 94,000 and 43,000 peaks (Fig. 3)were probably constituents of group 1 proteins.

Antigenic comparisons. It was essential to dis-tinguish antigens of the outer membrane pro-teins from the closely associated 0 antigens.The identification of antigens as LPS and nativehapten was based on possession of essentialproperties matching those previously described(16, 39), including their presence in extracts ofhot phenol and TCA (Fig. 7a), the presence ofhapten in cytoplasm and cell wall (Fig. 7b),hydrolysis of LPS into hapten (Fig. 7c), thepresence of antibodies for hapten in sera of fieldstrain-infected but not strain 19-vaccinated cat-tle (Fig. 7d), and enhanced development ofimmunoprecipitates of hapten in cattle sera with10% salt agar (data not shown).Immunodiffusion reactions were performed

on purified proteins of the five strains withabsorbed antisera specific for group 2 proteins ofstrains 19, 45/20, 2308, and Y and for group 3proteins of strains 45/20 and Y. Each system wasarranged to provide complete information oncross-reactions of heterologous with homolo-gous antigens (Fig. 8). Group 2 proteins devel-oped two distinct lines which in each of thereciprocal test systems were shared in an identi-cal way among all the strains (Fig. 8a and b).Before absorption, 0 determinants were alwaysassociated with the line closer to the serum well.Group 3 proteins produced a heavy broad bandclose to the serum well which could be resolvedinto two lines when antigens were diluted. Reac-tions of identity among the five strains werenoted with antisera specific for strains Y (Fig. 8cand d) and 45/20. In both antigen systems, one ormore additional faint lines, not uniformly sharedamong strains, were observed with some anti-sera. Group 2 and 3 antigens were immunologi-cally unrelated on the basis of immunodiffusionand immunoelectrophoresis (Fig. 6a and b). Serafrom 11 cattle infected with field strains of B.abortus, after absorption of 0 antibodies, failedto react with group 2 or 3 proteins in immunodif-fusion.

DISCUSSIONThe weight of evidence favors the hypothesis

that group 2 and 3 proteins of B. abortus areanalogous to matrix porins and OmpA, respec-tively. Group 2 proteins had a molecular weightin their denatured form comparable to mostporins, and their amino acid composition boreconsiderable resemblance to OmpF of E. coli(Table 4) and to porins of other gram-negativebacteria (27, 28, 45), although this in itself would

not ensure identity (45). Differential migration ofthese proteins after heating suggests their exis-tence as trimers in the native state, in accordwith observations on porins from other genera(19, 42, 45, 46, 59). Group 3 proteins were more

a_ _

c 40

CX

d

FIG. 6. Immunoelectrophoresis of B. abortus anti-gens with anode to right. (a) Upper and lower wells,strain 19 TCA extract; middle well, strain Y-purifiedgroup 2 proteins. Upper trough, rabbit antiserumspecific for strain Y group 2; lower trough, sameantiserum before absorption of 0 antibodies. (b) Up-per and lower wells, strain 19 TCA extract; middlewell, strain Y-purified group 3 proteins. Upper trough,rabbit antiserum specific for strain Y group 3; lowertrough, same antiserum before absorption of 0 anti-bodies. In (a) and (b), unabsorbed sera form precipi-tates with 0 antigens, but absorbed sera do not. Theprecipitate in the upper sector of (b) formed as a resultof migration of unabsorbed serum from (a), lowertrough, which was adjacent. Group 2 and 3 antigensproduce no precipitates comparable to the TCA ex-tract, indicating lack of unbound LPS, but precipitatesof the proteins with absorbed sera are lighter, suggest-ing physical association of 0 antigens with group 2 and3 proteins. The anodal streaks with protein antigenswere consistently observed with rabbit antisera andare considered artifacts. (c) Upper well, strain 19 TCAextract; middle well, strain Y-purified group 2 pro-teins; lower well, strain 19-purified group 2 proteinsfrom cells extracted with TCA before disruption.Troughs, antiserum from a cow with brucellosis. (d)Upper well, strain 19 TCA extract: middle well, strain2308-purified group 3 proteins; lower well, strain 2308-purified group 3 proteins from cells extracted withTCA before disruption. Troughs, same serum as in (d).Absorption experiments indicated that immunopreci-pitates produced by bovine sera with group 2 and 3proteins resulted from 0 determinants bound to theproteins (see text).

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TABLE 4. Amino acid compositions of group 2 and 3 proteins from B. abortus and OmpF and OmpA ofE. coli

Moles percentAmino acidE. colP OmpF B. abortush group 2 E. colt OmpA B. abortusb group 3

Asxc 17.4 11.7 ± 0.4 12.6 11.4 ± 0.7Thr 6.2 7.7 ± 0.3 6.5 5.5 ± 0.4*dSer 4.7 5.9 ± 0.2 4.9 4.6 ± 0.4*Glxc 7.1 9.4 ± 0.3 8.9 9.8 ± 0.7Pro 1.2 2.4 ± 0.4 5.8 4.2 ± 0.2*Gly 13.5 15.4 ± 0.5 11.4 14.5 ± 0.4*Ala 8.5 10.8 ± 0.2 8.9 10.2 ± 0.8Cys 0 ND' 0.6 NDVal 7.1 8.1 ± 0.6 7.7 6.7 ± 0.6*Met 0.9 0.6 ± 0.3 1.5 0.8 ± 0.7Ile 3.5 4.0 ± 0.6 4.3 6.7 ± 3.1Leu 6.2 5.3 ± 0.5 6.8 7.2 ± 0.2*Tyr 8.5 6.1 ± 0.1 5.2 3.2 ± 2.3*Phe 5.6 4.5 ± 0.1 2.5 3.4 ± 0.3*His 0.3 1.2 ± 0.2 1.5 2.2 ± 1.6Lys 5.3 4.0 ± 0.3 5.2 6.7 ± 0.6*Arg 3.2 3.8 ± 0.5 4.0 3.4 ± 0.3Trp 0.9 ND 1.5 NDa Derived from data in references 10 and 11.b Data are means ± SD for the five strains tested.c Asx, Aspartic acid and asparagine; Glx, glutamic acid and glutamine.d *Significantly different (P < 0.05) from value of corresponding residue in group 2 proteins.e ND, Not determined.

closely related in amino acid composition toOmpA than OmpF and differed from group 2proteins at some of the same residues as those atwhich OmpA differs from OmpF (10, 11). Group3 proteins were more refractory than were thoseof group 2 to detergent extraction. If this wereowing to their stronger association with murein,it would compare with OmpA, now believed tointeract more extensively than matrix porinswith murein (45). The migration of group 3proteins in SDS-PAGE after 100°C heating wascomparable to OmpA, but unlike homologs ofOmpA in other genera (4), heat-modifiable be-havior of group 3 proteins did not occur. Heatmodifiability of E. coli OmpA (51) and of severalproteins of Pseudomonas aeruginosa exclusiveof porins (21) was obviated by addition of LPS,and the LPS bound to each of our group 3proteins may have had a similar effect. In con-trast, the minor group 1 proteins, most of whichwere physically associated with group 3 unlessin SDS, exhibited both heat modifiability andapparent disaggregation at higher temperatures.

Effective solubilization of B. abortus outermembrane proteins, in particular, those of group3, was achieved only after digestion with lyso-zyme. The mechanism of lysozyme enhance-ment is uncertain. Cleavage of murein may haveweakened its association with protein or allowedbetter access of detergents. The requirement forlysozyme digestion was probably owing, at leastin part, to the use of Formalin-killed cells.

Zwittergent extraction of outer envelopes fromliving and Formalin-killed B. abortus could notbe compared owing to the risk of infection inpreparing envelopes from living B. abortus. Acomparison of this type was conducted with E.coli B and it was found that lysozyme treatmentdid not enhance Zwittergent extraction of outermembranes prepared from living cells but in-creased fourfold the percentage of protein ex-tracted from outer membranes derived fromFormalin-killed cells (D. R. Verstreate and A. J.Winter, unpublished data).

It is uncertain whether multiple banding pat-terns within each of the protein groups on SDS-PAGE reflect the existence of distinct proteinspecies. It is possible, for example, that the twoprincipal precipitin lines which developed withgroup 2 proteins were produced by antigenicallydistinct 43,000 and 41,000 proteins, but no directevidence for such an association is yet available.Multiple banding patterns have been observed inthe porins of a variety of gram-negative bacteria(15, 21, 34, 41, 45), but in only a few genera hasit been established that these represent separategene products (45). Multiple bands from a singleprotein species have been ascribed to severalfactors (28, 33, 48), and the variable number ofbands observed, for example, in group 3 pro-teins, among and within strains might be ex-plained by varying quantities of adherent mureinor LPS. This question in regard to the B. abortusproteins remains open.

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OUTER MEMBRANE PROTEINS OF B. ABORTUS

In none of our preparations was it possible tovisualize a low-molecular-weight protein compa-rable to the lipoprotein abundant in the outermembrane of many gram-negative bacteria (6,45). In contrast to P. aeruginosa (21, 22), lyso-zyme predigestion did not facilitate solubiliza-tion of lipoprotein and the addition of Mg2+ tosolubilized samples did not allow visualizationof a low-molecular-weight band. The presenceof lipoprotein covalently linked to murein couldnot be determined by amino acid analysis owingto the presence of other proteins on the mureineven after exhaustive boiling in SDS of eitherFormalin-killed (Fig. 2e) or living cells. Suchnegative data are of course not sufficient toexclude the occurrence of a lipoprotein in the B.abortus wall.Group 2 and 3 proteins were antigenically

distinct, as are the porins and heat-modifiableproteins of E. coli (23) and Neisseria gonorr-hoeae (25). In contrast to Neisseria spp. (7, 18,24) group 2 and 3 antigens were shared amongfive random strains, but more strains must betested to ensure that these are species-wideantigens. Production of antibodies to these pro-

FIG. 7. Immunodiffusion reactions of B. abortus 0antigens developed with sera from cows with brucello-sis. Agar contains 1%o NaCl. (a) Wells 1 and 2,antiserum; well 3, fraction 5, derived from phenolextraction of strain 19; well 4, acid-soluble phase fromTCA extract of strain 19; well 5, anodally migratingfraction derived from hydroxylapatite fractionation ofstrain 19 outer membrane proteins. Lines are LPS(closer to antigen well) and native hapten (29). (b) TCAextracts of whole cells (well 1) and cytoplasm (wells 5and 6) of strain 19 and whole cells (well 2) andcytoplasm (wells 3 and 4) of strain 2308. Serum incenter well. (c) TCA extract of strain 19 untreated(well 1) and hydrolyzed at 100°C in 1% acetic acid for5, 15, 30, 60, and 120 min (wells 2 through 6, respec-tively). Serum in center well. (d) Wells 1, 2, and 3, serafrom individual cows with brucellosis; wells 4, 5, and6, pooled sera from heifers vaccinated with strain 19.Center well, TCA extract of strain 19. The inner LPSline is only faintly visible in the serum pool of vacci-nated animals.

a

19 4520 2308

C

19 Y 4520

b

C10 4520 Y

d

CO V 2308

FIG. 8. Immunodiffusion reactions of purified out-er membrane proteins with specific rabbit antiserafreed of 0 antibodies by absorption. (a) and (b),bottom wells, group 2 proteins (2 mg/ml) of designatedstrains; top wells, antiserum specific for group 2proteins of strain 45/20. Split in strain Y precipitin lineis an artifact owing to an edge effect. (c) and (d),bottom wells, group 3 proteins (250 sLg/ml) of designat-ed strains; top wells, antiserum specific for group 3proteins of strain Y. One band with strain C-10 devel-oped very faintly but could be visualized when anti-gens were more concentrated.

teins in rabbits proved difficult owing to thedominant response to LPS. It is likely that theproteins functioned in some measure as carriersfor the tightly associated LPS determinants.Recent experiments have demonstrated thatproteins can be almost completely separatedfrom LPS by brief sonication in Zwittergentfollowed by TCA precipitation (D. R. Ver-streate, unpublished data). The antibody re-sponse in naturally infected cattle is known to bedirected predominantly to LPS and native hap-ten (16, 39), and in single samples from a fewanimals, we found no precipitins to group 2 and3 proteins. Responses to these proteins in vacci-nated and infected cattle are being evaluatedcurrently by enzyme-linked immunosorbent as-say and blastogenesis assays (C. L. Baldwin,unpublished data). Preliminary data indicate thatthe antigenic relatedness of these proteins be-tween vaccine strains and field strains does notlimit their usefulness in differentiating vaccinalresponses from clinical disease, and their role inconferring protective immunity is currently un-der investigation.

ACKNOWLEDGMENTSWe thank C. Fulimer for performing the amino acid analy-

ses, G. Barker for photography, and J. Reyna for preparingthe manuscript.

This research was supported in part by U.S. Dept. ofAgriculture grant 59-2361.-02-080-0.

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