INFECTION AND IMMUNITY, Nov. 1994, p. 5075-5084 Vol. 62, No. 110019-9567/94/$04.00+0Copyright C 1994, American Society for Microbiology

A Family of Phase- and Size-Variant Membrane SurfaceLipoprotein Antigens (Vsps) of Mycoplasma bovis

ANNET[ BEHRENS,1 MARTIN HELLER,2 HELGA KIRCHHOFF,' DAVID YOGEV,3AND RENATE ROSENGARTENl 3*

Institut fiir Mikrobiologie und Tierseuchen, Tierarztliche Hochschule Hannover, Bischofsholer Damm 15,30173 Hannover, 1 and Bundesinstitut fiir Gesundheitlichen Verbrauerschutz und Veterinarmedizin,

07743 Jena,2 Germany, and Department ofMembrane and Ultrastructure Research,The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel3

Received 30 December 1993/Returned for modification 18 April 1994/Accepted 8 August 1994

A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasmabovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family ofantigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). Bysystematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this familyhave been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequencychanges in size as well as noncoordinate phase variation between ON and OFF expression states. Themonoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface inmost, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragmentof each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished fromVspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibodyagainst M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by theircharacteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structuralfingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vspproduct, they showed distinct differences among variants differing in size. This variability included gain or lossof individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- ordownwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locationseither within the C-terminal domain or within other regions of these proteins. This was similarly confirmed bycomparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants.The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenicvariation in mycoplasmas displaying features which closely resemble but are also different from thecharacteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineagesestablished here provide key populations for subsequent analysis of corresponding genes to further elucidateVsp structure and variation, which may have important relevance for a better understanding of thepathogenicity of this agent.

The small wall-less prokaryotic species Mycoplasma bovisincludes both pathogenic organisms that cause mastitis, arthri-tis, pneumonia, and diseases of the genital tract in the naturalbovine host (11, 12, 20) and harmless commensals that areoccasionally isolated from the respiratory tract and from milksamples of clinically healthy animals (2, 12, 20). To understandthese differences in pathogenic potential among M bovisisolates, it is important to understand the molecular mecha-nisms and population dynamics of phenotypic variation, par-ticularly those associated with the membrane surface structureof this organism. Because of the direct interaction of themycoplasma membrane surface with host cells and the hostimmune response, structural and antigenic differences ex-pressed on that surface are becoming increasingly appreciatedas playing a central role in dictating several phenotypicallyvariable characteristics of pathogenic mycoplasmas, includingvirulence features (6, 14, 19, 29, 30, 31). Although the precisenature and extent of phenotypic diversity in the host have yetto be determined for any infectious mycoplasma species,

* Corresponding author. Present address: Department of Mem-brane and Ultrastructure Research, The Hebrew University-HadassahMedical School, P.O. Box 12272, Jerusalem 91120, Israel. Phone: (972)2-758176. Fax: (972) 2-757413.

molecular surface features and the actual parameters creatingand maintaining diversity have recently come to light in somespecies by analysis of the phenotypic dynamics of populationsin vitro (14, 18, 23-27, 31, 37). Detailed assessment of onediversity-generating system in Mycoplasma hyorhinis, a swinepathogen, has revealed a unique strategy for surface diversifi-cation through a set of variable membrane surface lipoproteins(Vlps) which undergo high-frequency changes in size andexpression (5, 23-25, 32, 35, 37). Recent work has indicatedthat other pathogenic mycoplasma species may possess similarstrategies for diversification (16, 17, 26, 27, 34; reviewed inreference 35). One such species is M. bovis, which was shown inthe preceding paper (22) to greatly vary among strains andwithin clonal lineages of the PG45 strain in the expression andsize of multiple surface-exposed membrane surface proteinantigens, which were found to be major targets of the hostimmune response during disease caused by this agent.

In the present study, we further investigate the antigenic andstructural features of these variable membrane proteins anddefine the precise parameters contributing to heterogeneity inM. bovis surface architecture. Using an enzyme-based methodof protein fingerprinting and immunological reagents previ-ously developed to identify M. bovis variable antigens, as wellas metabolic labeling and protein staining techniques, we

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define a set of lipid-modified, antigenically and structurallyrelated but distinct translation products making up the Vsp(variable surface protein) family of this organism. We furtherdemonstrate that this system shares several characteristics withthe Vlp system of M. hyorhinis.

MATERIALS AND METHODS

Mycoplasmas, cultivation, and harvesting. M. bovis typestrain PG45 originated from the collection of the Institute ofMicrobiology and Infectious Diseases of Animals, School ofVeterinary Medicine, Hannover, Germany, as a filter-clonedculture and was grown at 37°C in a previously describedstandard mycoplasma broth medium (22) containing 20%(vol/vol) heat-inactivated horse serum. Stocks of strain PG45were prepared by freezing 1-ml aliquots of a 10-ml logarithmic-phase broth culture at -80°C. For subcloning, frozen stockswere thawed, and 100-,ul aliquots were expanded in 1-ml brothcultures before being plated on 1% agar medium. Clonallineages were generated as previously described (22). Forradiolabeling, detergent-phase fractionation, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), andWestern blotting (immunoblotting), organisms were harvestedfrom logarithmic-phase cultures by centrifugation for 5 min at12,000 x g, washed twice in phosphate-buffered saline (PBS;2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4, pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluo-ride (Fluka, Neu-Ulm, Germany), and processed as describedbelow.

Radiolabeling of mycoplasmas. M. bovis PG45 subcloneswere metabolically labeled with L-[355]cysteine or [9,10-3H]palmitic acid, using methods described elsewhere (25), withslight modifications. Briefly, 1.8 ml of broth cultures containingapproximately 2.6 x 108 CFU was centrifuged for 5 min at12,000 x g. The cell pellets were washed once with PBS-phenylmethylsulfonyl fluoride, suspended in 40 [LI of mediumcontaining the appropriate label (see below), and incubated for18 h at 37°C. The labeled cells were harvested by centrifugationas described above, washed twice with ice-cold PBS-phenyl-methylsulfonyl fluoride, and subjected to Triton X-114 (TX-114) phase fractionation as described below.For metabolic labeling with cysteine, cysteine-free medium

was prepared by dialyzing complete mycoplasma broth me-dium against several changes of cysteine-free RPMI 1640(Gibco BRL, Eggenstein, Germany). For labeling, the dialyzedmedium was supplemented with 0.8 mCi of L-[355]cysteine(specific activity, 1,220 Ci/mmol; Du Pont de Nemours, BadHomburg, Germany) per ml.For metabolic labeling with palmitic acid, an ethanol solu-

tion containing [9,10-3H]palmitic acid (specific activity, 60Ci/mmol; Du Pont de Nemours) was dried under air andredissolved in a small volume of ethanol, which was added tomycoplasma growth medium in a concentration of 2% (vol/vol)to give 1 mCi of [3H]palmitic acid per ml.

Antibodies. The preparation and characteristics of antibod-ies used in this study have been previously described in detail(3, 22). Monoclonal antibody (MAb) 1E5, which was raisedagainst a clonal variant of M. bovis PG45, is an immunoglob-ulin M(K) isotype. Previously described as a MAb recognizinga set of strain- and size-variant membrane surface proteins(22), it is shown here to react with VspA, VspB, and VspC. Arabbit polyclonal antibody (PAb) against M. bovis D490, whichwas used in the preceding paper (22) to initially detectdifferences in the expression and size of multiple antigensamong and within strains of M. bovis, reacts with VspA andVspC. The construction and characteristics of MAb 5D8 have

been described elsewhere (3). It is an immunoglobulin G2a(K)isotype (3) recognizing p41, a 41-kDa internal protein (22). Foruse in colony immunoblotting, serum and hybridoma culturesupernatants containing MAbs were diluted in PBS; for use inWestern immunoblotting, they were diluted in PBS containing0.1% (vol/vol) Tween 20 (Merck, Darmstadt, Germany) (PBS-T).Mycoplasma protein analysis. TX-114 (Boehringer Mann-

heim, Mannheim, Germany) phase fractionation of M. bovisPG45 subclones was carried out as described in the precedingpaper (22), using unlabeled or metabolically labeled organ-isms. Total or TX-114 phase-fractionated proteins were sepa-rated by SDS-PAGE as previously described (22). After elec-trophoresis, gels were stained with silver by the procedure ofBlum et al. (4). Gels containing radiolabeled proteins werefluorographed as described in detail elsewhere (7, 33). Driedgels were exposed at -800C to X-Omat-AR films (Kodak,Rochester, N.Y.) for 2 to 8 weeks (35S) or up to 16 weeks (3H).Nitrocellulose membrane filter blots were prepared by themethod of Towbin et al. (28) and either stained with colloidalgold with the Enhancement Colloidal Gold Total ProteinDetection Kit (Bio-Rad, Munich, Germany) or immunostainedas previously described (22), using MAbs lE5 (if not specifi-cally indicated, diluted 1:100 in PBS-T) and 5D8 (undiluted)and rabbit antiserum to M. bovis D490 (diluted 1:1,000 inPBS-T) as primary antibodies and peroxidase-conjugated goatantisera (Nordic; diluted 1:1,000 in PBS-T) to rabbit immuno-globulins, mouse immunoglobulin G (heavy and light chains),or mouse immunoglobulin M (Fc fragment) for detection.Blots containing [35S]cysteine-labeled proteins were immuno-stained, dried, and autoradiographed for 10 weeks by directexposure to films at - 80°C. For staining proteins with colloidalgold, blots were dried and incubated for 30 min at 60°C andthen stained (without blocking) for 2.5 h in staining solutionsupplied with the kit, according to the manufacturer's recom-mendations.

Carboxypeptidase treatment of intact mycoplasmas. Treat-ment of intact organisms with carboxypeptidase has beendescribed elsewhere (24). Briefly, organisms were harvested bycentrifugation for 5 min at 12,000 x g from logarithmic-phasebroth cultures, and cell pellets were rinsed once with PBS atpH 7.2 and once with PBS at pH 6.0. Identical portionscontaining washed organisms from 100 p1 of broth culturewere centrifuged, and the mycoplasmas were resuspended inPBS (pH 6.0) containing either no enzyme or various concen-trations of carboxypeptidase Y (Pierce, Oud-Beijerland, TheNetherlands). After 2 h at 37°C, SDS-PAGE sample buffer wasadded, and the samples were heated for 3 min at 100°C andsubjected to SDS-PAGE. Subsequent immunoblots wereprobed with MAb 1E5 (diluted 1:10 in PBS-T) as describedabove. In some experiments, blots were restained with MAb5D8 to demonstrate cell integrity during digestion.

Trypsin treatment of intact mycoplasmas. Intact organismswere treated with trypsin as previously described (22). Westernblots of SDS-PAGE-separated digestion products were pre-pared and immunostained with the MAb and PAb reagents asdescribed above. To demonstrate the physical separation of aC-terminal MAb-defined and an N-terminal PAb-defined tryp-tic fragment of a Vsp, similar preparations containing trypsin-treated mycoplasmas were subjected to TX-114 phase fraction-ation as described before (22). Prior to solubilization, soybeantrypsin inhibitor (Sigma, St. Louis, Mo.) was added to eachenzyme reaction mixture to a final concentration of 1 ,ug/,u.

Colony immunoblotting. Vsp phase variants were detectedby colony immunoblotting with MAb 1E5. Colony blots were

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FIG. 1. Antigenic and structural signatures of VspA, VspB, and VspC. (a and b) Whole organisms from clonal variants of M. bovis PG45expressing a single VspC (lane 1), VspA (lane 2), or VspB (lane 3) size variant were subjected to SDS-PAGE and immunoblotted with MAb lE5(a) or with rabbit antiserum to whole broth-grown organisms from M. bovis D490 (b), as previously described (22). Each sample contained proteinsfrom approximately 1.5 x 106 (a) or 3 x 106 (b) CFU. VspA, VspB, and VspC are indicated as the corresponding lettered bands A, B, and C. Thesize of each Vsp variant calculated from prestained standard markers run in the same gel is indicated in kilodaltons. (c to e) Intact organisms(approximately 3.5 x 106 CFU per sample) from clonal variants (corresponding exactly to those in panels a and b) incubated without enzyme orwith increasing concentrations of carboxypeptidase Y were subjected to SDS-PAGE, and the resulting immunoblots were stained with MAb 1E5,as described in Materials and Methods. Lanes 1 contain untreated organisms, and lanes 2 through 6 contain organisms treated, respectively, with40, 100, 150, 200, or 400 ,ug of carboxypeptidase Y per ml. Positons and molecular masses (in kilodaltons) of the intact VspC (c), VspA (d), andVspB (e) and of their very smallest immunostained digestion products are indicated. VspA-, VspB-, or VspC-characteristic arrays of bands withineach ladder of epitope-bearing C-terminal truncation products (lanes 2) are indicated by brackets. A characteristic narrow-spaced doublet isindicated by an asterisk. Restaining of blots with MAb 5D8 to internal p41 protein showed this protein to be unaffected by external treatment withenzyme, thereby indicating that cells remained intact during digestion (data not shown).

obtained and immunostained as previously described in detail(22).

RESULTS

Identification and characterization of distinct Vsps. In thepreceding paper, we have described a set of prominent size-variant membrane surface proteins which were detected inclonal populations ofM bovis PG45 by both a specific MAbraised against a clonal variant of that strain and a rabbitantiserum raised against M bovis D490 (22). One protein, a46-kDa variant of this set, however, was not recognized by thePAb reagent. This finding, which is confirmed in Fig. la and bfor other variant clones of the PG45 strain, suggested that atleast two distinct translation products may exist among thesevariable surface proteins (Vsps). To further define individualVsps, epitope mapping studies with the MAb were performedto identify possible differences within the internal protein

structure which would allow us to distinguish between distinctproducts. This was accomplished by using a recently developedprocedure that truncates proteins on the surface of intactmycoplasma cells by graded digestion with carboxypeptidase Y(24), a protease that sequentially cleaves amino acids from theC-terminal end of proteins and hydrolyzes some amino acidresidues (e.g., Gly, His, Arg, and Lys) at a much reducedefficiency (9). Results of this epitope mapping analysis of 77-,65-, and 46-kDa Vsp variants are shown in Fig. lc to e. Partialdigestion of these variants revealed a characteristic ladder ofepitope-bearing, C-terminally truncated products (Fig. lc to e,lanes 2) with a minimum size of 50 (Fig. lc), 40 (Fig. ld), or 28(Fig. le) kDa and an irregular, sometimes periodic spacing,suggesting a corresponding repetitive spacing of carboxypepti-dase pause sites within or between distinct structural units. Inmore extensively digested preparations, these ladders gradu-ally disappeared (Fig. lc to e, lanes 3 to 6). By comparing their

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FIG. 2. Detergent-phase fractionation, metabolic labeling, and staining characteristics of VspA, VspB, and VspC. Clonal variants of M. bovisPG45 expressing a single size variant of VspC (lanes 1), VspA (lanes 2), or VspB (lanes 3) are shown. Identical populations are represented inthe corresponding lanes of each panel. The identity of each Vsp was confirmed as described in the legend to Fig. 1. Their relative molecular massescalculated from standard markers are indicated in kilodaltons. (a) Whole organisms were subjected to TX-114 phase fractionation, and proteinsfrom total organisms (T) or the TX-114 (TX) or aqueous (Aq) phase were separated by SDS-PAGE and electrophoretically transferred tonitrocellulose, as described in Materials and Methods. After immunostaining with MAb lE5, the blot was restained with MAb 5D8 to internalhydrophilic protein p41. The position and size (in kilodaltons) of this protein are indicated. Samples contained proteins from approximately 6 x106 (T and Aq) or 108 (TX) CFU. VspA, VspB, and VspC are indicated by lettered bands A, B, and C. (b and c) Fresh broth-grown organismsmetabolically labeled with [3H]palmitate (b) or [35S]cysteine (c) were phase fractionated, and proteins from TX-114 phases were separated bySDS-PAGE and visualized by fluorography, as described in Materials and Methods. Each lane represents phase-fractionated proteins fromapproximately 2 x 108 CFU. (d and e) Whole organisms were subjected to SDS-PAGE and either stained with silver (e) or electrophoreticallytransferred to nitrocellulose and stained with colloidal gold (d), as described in Materials and Methods. Each sample contained proteins fromapproximately 2 x 10i CFU.

antigenic (Fig. la and b) and structural (Fig. lc to e) profiles,the 77- and 65-kDa Vsp variants appeared to be more closelyrelated to one another than to the 46-kDa Vsp variant.However, although the ladders generated from the 77- and65-kDa variants showed striking similarities, reproducible dif-ferences were also evident (Fig. lc and d). For example, thespacing between the four lower bands was greater in the77-kDa variant than in the 65-kDa variant. In addition, thesmallest epitope-bearing truncation product differed signifi-cantly in size (50 versus 40 kDa). Taken together, the structuralfingerprints obtained by this method were distinctive for eachof the three proteins, indicating structural differences amongthem. Thus, on the basis of the immunoprofiles obtained withthe MAb and PAb reagents (Fig. la and b) in combination withthe structural fingerprints obtained with carboxypeptidase Y,we were able to identify three distinct Vsp translation productswhich constitute the Vsp protein family. These were desig-nated VspA (Fig. la and b, lanes 2; Fig. ld), VspB (Fig. la andb, lanes 3; Fig. le), and VspC (Fig. la and b, lanes 1; Fig. lc).Furthermore, the experiments described above clearly demon-strated that the epitope recognized by the MAb was distinctfrom those detected by the PAb (Fig. la and b) and waslocated on a C-terminally truncated product with a minimumsize of 50 (VspC; Fig. lc), 40 (VspA; Fig. ld), or 28 (VspB; Fig.le) kDa. The presence of the undegraded internal p41 proteinin the extensively digested preparations (not shown) furtherindicated that the cells remained intact during treatment withthe enzyme. Collectively, these results suggested that each Vspis anchored in the membrane by N-terminal regions while mostof its C-terminal portion is fully accessible at the externalsurface of the organism.

Besides its distinctive antigenic and structural characteris-tics, VspB was further shown to be distinguishable from VspAand VspB by its lack of staining with colloidal gold (Fig. 2d).

Despite these differences among them, VspA, VspB, and VspCwere shown to have several common properties. First, theywere shown to be amphiphilic integral membrane proteins. Aspreviously described (22), this was demonstrated by theirexclusive partitioning into the detergent phase during TX-114phase fractionation (Fig. 2a). Second, each Vsp was shown byfluorography of SDS-PAGE gels (Fig. 2b and c) or autoradiog-raphy of Western immunoblots (not shown) to be metaboli-cally labeled with [3H]palmitate (Fig. 2b) and [35S]cysteine(Fig. 2c). Third, VspA, VspB, and VspC were refractory tostaining with silver (Fig. 2e). These results, notably, the am-phiphilic character, the presence of at least one Cys residue,and the presence of fatty acids, clearly identified VspA, VspB,and VspC as putative lipoproteins. In addition, the intensitiesof the autoradiographic signals of VspA, VspB, and VspC from[3H]palmitate- or [ 5S]cysteine-labeled mycoplasma cells rela-tive to those of other TX-114 phase proteins (Fig. 2b and c)and the intensities of immunostaining of VspA, VspB, andVspC with the MAb (e.g., Fig. la and 2a) and of VspA andVspC with the PAb (Fig. lb) argued that Vsps are predomi-nant, if not the most abundant membrane proteins of theorganism. Moreover, having established that VspA, VspB, andVspC were discrete proteins which were identified by theirdistinctive epitope profiles and structural fingerprints, we wereable to detect two key properties of these products which aredescribed in the following three sections: (i) high-frequencysize variation (Fig. 3, 4, 5, and 6), and (ii) high-frequency phasevariation in expression (Fig. 6).

Size variation of Vsps. So far, we have documented 10different individual size variants of VspA, 9 of VspB, and 5 ofVspC, 9 of which (i.e., 3 variants of each Vsp) are shown in Fig.3a. Since all samples were prepared in the presence of proteaseinhibitor, these differences in the size of the expressed Vspamong clonal populations which were shown to be stable in

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FIG. 3. High-frequency size variation of VspA, VspB, and VspC within a clonal lineage of M. bovis PG45. (a) Whole organisms from selectedclonal variants expressing individual size variants of VspC (lanes 1 to 3), VspA (lanes 4 to 6), or VspB (lanes 7 to 9) were subjected to SDS-PAGEand immunoblotted with MAb 1E5. Immunoblots were prepared as described in the legend to Fig. 1; each channel was loaded with proteins fromapproximately 106 CFU. VspA, VspB, and VspC are indicated by lettered bands A, B, and C; their assignment was based on lack of immunostainingof VspB by rabbit antiserum to strain D490 and on specific structural fingerprints generated by carboxypeptidase Y, as described in the legend toFig. 1 (data not shown). Molecular masses of the very largest and the very smallest VspA, VspB, and VspC size variants shown are indicated inkilodaltons. (b to d) Immunoblots of individual randomly selected progeny populations (siblings) of three clonal variants (not shown), eachexpressing an individual size variant of VspC (b), VspA (c), or VspB (d), were prepared as for panel a. Samples contained approximately 2 x 106to 3 x 106 CFU. The molecular mass of the predominant Vsp size variant expressed within each group of siblings is indicated in kilodaltons. Theidentities of VspA, VspB, and VspC were confirmed as described for panel a (data not shown). The 63-kDa band in panel d represents a VspAsize variant which is expressed by minor switched subpopulations in the clonal populations selected for the predominant VspB phenotype.

multiple preparations could not be explained by general pro-teolytic degradation. To illustrate the rapidly and spontane-ously occurring changes in apparent Vsp size within clonalpopulations, a series of subclones which were randomly se-lected from the immediate progeny of three clonal variantsexpressing a 75-kDa size variant of VspC, a 65-kDa variant ofVspA, or a 43-kDa size form of VspB is shown in Fig. 3b, c, andd, respectively. None of the three clonal variants resulted inpopulations which were stable in the size of the expressed Vsp.In all three cases, two to six of the eight subclones clearlydiffered from the parental clone by a Vsp size shift to lower(panel b, lane 7; panel c, lanes 3 and 5; panel d, lanes 3, 4, and6) or higher (panel b, lanes 1 to 4, and 6) molecular weights.The rapidly generated changes in apparent molecular size ofVspA, VspB, or VspC were also reflected in the heterogeneousladder patterns observed in most of the subclones analyzed(Fig. 3b to d). These laddered sets of multiple Vsp size variantforms which were generated at a very high frequency withinthese subcloned populations were readily apparent in theimmunoblots shown in Fig. 3b to d, because two to three times

more material was loaded than normally used (Fig. 3a) tovisualize discrete Vsp size variants. The spontaneously occur-ring size variants which collectively accounted for the observedladder patterns showed either uniform or nonuniform spacingcorresponding to a difference in apparent molecular weight ofapproximately 500 to 2,000. This irregularity was most pro-nounced in VspB variants, as shown in Fig. 3d. Although it isnot clearly evident from Fig. 1 and 3, we could demonstrate inimmunoblots of several heavily loaded samples that the spon-taneously occurring ladders of Vsp size variants correspondedquite precisely to the laddered sets of partial carboxypeptidasetruncation products. This again argued for the presence ofrepetitive different-sized structural units within these proteins.

Identification and localization of structural changes gener-ating Vsp size variation. In an initial attempt to monitorstructural changes associated with Vsp size variation, we tookadvantage of the Vsp fingerprinting method, using carboxypep-tidase Y as described above. This strategy, which was appliedto both different- and similar-sized Vsp variants from severalisogenic lineages, allowed us to define quite precisely regions

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FIG. 4. Changes in protein structure during Vsp size variation. Fresh broth-grown organisms from clonal variants of M. bovis PG45 expressingindividual size variants of VspC (a), VspA (b), or VspB (c) were incubated with 40 ,ug of carboxypeptidase Y per ml and subjected to SDS-PAGEas described in Materials and Methods. The subsequent immunoblots were stained with MAb lE5 as described in the legend to Fig. 1. Each lanecontains proteins from approximately 5 x 106 CFU. The clonal variants in panel a, lanes 1, 3, and 4, were selected from the immediate progeny

of the variant in lane 2 and correspond to the variants in Fig. 3a, lanes 1, 2, and 3, respectively. The clonal variants in panel b, lanes 3 to 5, representsiblings derived from the same parent colony (not shown). The variants in lanes 1 and 2 of panel b are from other clonal lineages. The clonalvariants in panel c derived from the same parental clone and correspond to the variants in Fig. 3a, lanes 7 and 8, respectively. Positions andmolecular masses (in kilodaltons) of the intact Vsps and their very smallest immunostained truncation products are indicated as determined forFig. 1. Immunostaining of the 28-kDa VspB peptide (c) is faint and at the limit of visual detection. Variable regions involved in Vsp size variationare indicated by brackets.

within the Vsp molecules showing structural alterations duringVsp size variation. Some representative examples of thisfingerprint analysis are shown in Fig. 4. While the bandingpatterns generated by partial digestion with the enzyme wereidentical for similarly sized VspA (panel b, lanes 1 to 3), VspB(not shown), or VspC (panel a, lanes 2 and 3) variants (withoccasional exceptions; data not shown), they were clearlydifferent for variants differing in size (VspA, panel b; VspB,panel c; VspC, panel a). These differences were manifest as

gain or loss of individual bands within distinct subsets of bands(indicated by brackets in Fig. 4) and corresponded exactly tothe observed increase or decrease in Vsp size. In some cases,however, Vsp size variation was not accompanied by anydetectable changes within the fingerprint pattern but by cor-

responding expansion or contraction of each truncation prod-uct bearing the MAb-defined epitope. One example of suchconcomitant size shifts is shown in Fig. 4b between theidentical ladders of truncated products generated from twodifferent-sized isogenic VspA variants (lanes 3 and 5). Collec-tively, these findings identified the surface-exposed C-terminalportion of the Vsp molecule as the predominant proteindomain undergoing changes during Vsp size variation. Inaddition, the results indicated that similar changes contributingto Vsp size variation may also occasionally occur within otherregions of the proteins.These conclusions were further supported in separate exper-

iments by mapping the MAb- and PAb-defined epitopes totryptic fragments of the Vsp molecules. Partial digestion ofindividual size variants of VspA (Fig. 5b) or VspC (Fig. 5a)expressed on the surface of intact cells with trypsin (whichpredominantly cleaves at Lys and Arg residues) yielded twoimmunoreactive fragments which together constitute the com-plete Vsp molecule: (i) one larger fragment bearing theepitope recognized by the MAb and showing size variation (inFig. 5, ranging from 41 to 45 kDa) which exactly corresponded

to the observed increase or decrease in the size of the nativeVsp (Fig. 5a; Fig. Sb, lanes 1 and 2); and (ii) one smaller,size-invariant 34 (VspC; Fig. 5d)- or 22 (VspA; Fig. 5e)-kDafragment bearing the epitopes detected by the PAb (Fig. 5dand e). These results indicated that the larger MAb-bindingtryptic fragment of VspA and VspC contained the surface-exposed, size-variant, C-terminal portion, whereas the smallerPAb-binding fragment represented the size-invariant, N-termi-nal, membrane-anchored portion of these proteins. In thosecases, however, in which Vsp size variation was not associatedwith changes within the C-terminal region, the smaller trypticfragment instead of the larger one was shown to vary in size.One example is the 67-kDa VspA size variant shown in Fig. 4b,lane 5. By comparing the tryptic fragments of that variant (Fig.Sb and e, lanes 3) with those obtained from a smaller 65-kDavariant (Fig. 5b and e, lanes 1), it is clearly evident that the sizedifference among these two proteins is due to structuralalterations within the N-terminal region of the VspA molecule,which is apparent as a corresponding size difference of 2 kDabetween the two PAb-binding 22- and 24-kDa fragments (Fig.Se, lanes 1 and 3). That this smaller PAb-defined trypticfragment of VspA and VspC contained, in fact, the putativeN-terminal domain for membrane insertion was confirmed byadditional experiments. After incubation of the organisms withtrypsin as in Fig. Sa and b, the resulting digestion productswere subjected to TX-114 phase fractionation, and TX-114 andaqueous phases were applied to SDS-PAGE and analyzed inimmunoblots. While the smaller PAb-defined fragment re-

mained associated with the membrane after cleavage andtherefore partitioned exclusively into the detergent phase (Fig.Sf, lanes 1 to 3), the larger C-terminal fragment bearing theMAb-defined epitope was released from the membrane andpartitioned into the aqueous phase (Fig. Sf, lanes 4 to 6). Asexpected, a similar segregation of the two fragments was

observed after centrifugation of the trypsin-treated organisms.

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FIG. 5. Separation of a C-terminal, MAb-defined portion and an N-terminal, PAb-defined portion of Vsp size variants. (a to e) Intactbroth-cultured whole organisms (approximately 3 x 106 CFU per sample) from clonal variants of M. bovis PG45 expressing individual size variantsof VspC (a), VspA (b), or VspB (c) were incubated with trypsin at 5 (a and b) or 200 (c) ,ug/ml as previously described (22). The digestion reactionmixtures were immediately prepared for SDS-PAGE, and subsequent immunoblots were stained with MAb 1E5 (a to c). In panels d and e, theblots in panels a and b were restained with the PAb to M. bovis D490. The presence of the undegraded internal p41 protein immunostained withthe PAb reagent (d and e) ensured cell integrity during digestion. The absence of PAb-defined epitopes on the MAb-binding tryptic fragments wasconfirmed by separately immunostaining identical blots with the PAb. The clonal variants in panels a, lanes 1 to 3, and in panel b, lanes 1 to 3,correspond to the variants represented in Fig. 4a, lanes 1, 3, and 4, and Fig. 4b, lanes 1, 4, and 5, respectively. Positions and molecular masses ofthe undegraded Vsp variants (a to c, left) and their epitope-bearing tryptic fragments (a to e, right), as well as of p41 (d and e), are indicated.Additional unmarked bands which also faintly stained with the MAb (a to c) represent corresponding tryptic fragments generated from smaller,alternative Vsp size forms expressed by minor subpopulations. (f) Digestion products shown in panels a and d, lanes 2, and in panels b and e, lanes1 and 3, were subjected to TX-114 phase fractionation, and products from TX-114 detergent (lanes 1 to 3) and corresponding aqueous (lanes 4to 6) phases were separated by SDS-PAGE and visualized by MAb and PAb immunostaining as in panels d and e. Positions and molecular massesof the undegraded VspC (lane 1, left) and VspA (lanes 2 and 3, left) variants and their PAb-defined N-terminal (lanes 1 to 3, right) andMAb-defined C-terminal (lanes 4 to 6, right) tryptic fragments are indicated. The MAb-defined tryptic fragment of the VspB size variants shownin panel c similarily separated into the aqueous phase (data not shown). p41 (lanes 4 to 6) is indicated on the left. The quantity of material addedto SDS-PAGE was adjusted to allow clear visualization of the undegraded Vsp variants and their MAb- and PAb-defined tryptic fragments.

While the PAb-defined fragment was associated with the cellpellet, the MAb-bearing fragment was released into the super-natant (data not shown). Partial digestion of VspB size variantswith trypsin required a much higher concentration of theenzyme (200 instead of 5 ,ug/ml used for VspA and VspC) andyielded only one fragment that was detected in immunoblots.This fragment showed features demonstrated above to be alsocharacteristic of the larger tryptic fragment of VspA and VspC.First, it contained the epitope recognized by the MAb (Fig. 5c).Second, it was of similar size (in Fig. 5c, ranging from 41.5 to44.5 kDa) and showed a size polymorphism corresponding tothe observed size heterogeneity among the undegraded VspB

variants (Fig. Sc). And third, it was released from intact cellsand selectively partitioned into the aqueous phase duringTX-114 phase fractionation (data not shown). From theseresults, it was clear that the trypsin-sensitive site was locatedvery close to the N terminus of the VspB molecule, in adistance of approximately 1.5 kDa, and was therefore lessaccessible for the enzyme.

In a series of supplemental experiments with graded trypsindigestion of intact cells, most of the VspA, VspB, and VspCsize variants analyzed were progressively degraded to thetryptic fragments described above (Fig. 5) without goingthrough intermediate products. Exceptions to these character-

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FIG. 6. Independent phase and size variation of VspA and VspB in a clonal lineage of M. bovis PG45. (a) Phase variation between ON (lanes1 and 3) and OFF (lane 2) expression states of a VspA size variant. (b and c) Sequential independent switching of VspA and VspB involving eitherconcomitant (b, lanes 2>-*3--4) or independent (b, lanes 1-*2, 2-*3-->6, and 4--5; and c) occurrence of phase and size variation. Immunoblots ofwhole cells stained with MAb lE5 were prepared as described in the legend to Fig. 3. Each lane represents approximately 2 x 106 CFU. VspAand VspB in this clonal lineage were identified as described in the legends to Fig. 1 and 3 and are indicated by lettered bands (a) or by brackets(b and c). The size of each Vsp variant calculated from standard markers is indicated in kilodaltons. Phenotypic transitions are indicated by arrows

above each immunoblot. In some cases, siblings are bracketed. Restaining of the blots in panels a and b with the rabbit antiserum to M. bovis D490revealed in lanes 2 (a) and 3 (b) the presence of alternative membrane protein antigens lacking the lE5 epitope (data not shown).

istic cleavage patterns occasionally occurred. One example isthe partial cleavage pattern of the 67-kDa VspA variantdescribed above (Fig. 5b, lane 3) showing an additionalintermediate tryptic peptide of approximately 46 kDa identi-fied by the MAb. In these experiments using increasing con-centrations of trypsin, the MAb-binding C-terminal trypticfragment of each Vsp variant was further shown to be a limitdigestion product, since it was trypsin resistant.

Independent phase transitions and combinatorial expres-sion ofVsp variants. Another key feature of VspA, VspB, andVspC was phase variation in expression. In the precedingpaper, by immunostaining colonies of subcloned PG45 popu-lations with the MAb, we have initially demonstrated a markedinstability in the expression of the epitope recognized, manifestas striking differences in the distribution of that epitopebetween and within individual colonies (Fig. 4g in reference22). In the present study, several clonal lineages showinghigh-frequency phase variation in Vsp surface epitope expres-sion were established by this technique. This epitope switchingwas shown by Western immunoblot analysis to correlate withthe differential expression of the corresponding VspA, VspB,or VspC. In one example of this analysis, oscillating expressionof a 65-kDa VspA size variant (Fig. 6a) is shown to correspondprecisely to the epitope expression pattern obtained in colonyimmunoblot. In some lineages, however, surface epitope-negative, unstained clonal variants were detected, which inWestern immunoblots clearly showed the expression of dis-crete VspA or VspC size variants bearing the MAb-definedepitope (not shown). This indicated that in these populationsthe epitope was masked and therefore, in colony immunoblots,not accessible at the surface. In contrast, the epitope-negativeclonal variants represented in panels a (lane 2) and b (lane 3)were shown to express alternative amphiphilic membraneproteins which could not (by the criteria described above) becategorized as VspA, VspB, or VspC. These alternative prod-ucts which were detected by the PAb (not shown) but lackedthe epitope recognized by the MAb have been previouslyclassified as antigen cluster I (22). In another case, epitope-negative clonal populations which completely lacked all threeVsps, as well as all other variable membrane protein antigenswhich could be identified by the PAb, were found.By monitoring the Vsp expression profiles in several lin-

eages, we could further demonstrate the following features ofVsp phase variation (Fig. 6). First, phase variation of a givenVsp occurred either independently (VspA, panel a; VspB,panel b, lanes 2->3-->6) or concomitantly (VspB, panel b, lanes2->3-4) with size variation in that protein. Second, althoughback-switch subpopulations expressing alternative Vsps werefrequently detected as minor bands in the broth-culturedpopulations analyzed (e.g., VspA in Fig. 2a, lanes 3; and Fig.3d), it seemed that any one Vsp could be coexpressed with anyother one or two Vsps (e.g., VspA plus VspB in Fig. 6b, lane2; and Fig. 6c, lanes 1 to 3). And third, phase transitionsinvolving multiple Vsps were not coordinately linked andoccurred with each Vsp switching independently from oneanother (e.g., VspA and VspB in Fig. 6b and c). Takentogether, these findings indicated that an individual M. boviscell can give rise to a large number of variant cell lines, eachexpressing distinct Vsp surface mosaics with different struc-tural and antigenic characteristics. Although immunologicalreagents specific for each Vsp were not available to clearlydemonstrate the coexpression of Vsps by replicate colony blotsor by immunoelectron microscopy, the following observationsstrongly argued that Vsps can in fact be expressed as mosaics.First, clonal variants which expressed multiple Vsps simulta-neously (e.g., VspA plus VspB in Fig. 6c, lane 1) could beclearly distinguished from those populations which were "con-taminated" by back-switch variants. While the coexpressionpatterns remained relatively stable in subsequent generations(e.g., VspA plus VspB in Fig. 6c, lanes 1 to 3), the level ofdetection and degree of contamination by switched subpopu-lations depended on (i) the frequency of Vsp switching,ranging from 10-3 to 10-' per cell per generation; and (ii) theamount of proteins loaded per sample. As shown in Fig. 3d,samples containing proteins from approximately 2 x 106 to 3 x106 organisms were sufficient to detect such contaminatingback-switch populations (in Fig. 3d, represented by the minor63-kDa VspA size variant). Second, we have recently identifieda cloned genomic fragment of the clonal variant shown in Fig.6c, lane 1, with the coexpression phenotype VspA (63 kDa)+VspB (46 kDa)+ VspC- (21, 36). Expression of this fragmentin Escherichia coli revealed two distinct translational productswith the size and epitope profile of the authentic VspA andVspB, respectively (36). From the finding that the correspond-

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ing vsp genes can be coexpressed in E. coli from a clonedgenomic fragment, we can conclude that Vsps can indeed beexpressed simultaneously by a single M. bovis organism.

DISCUSSION

From intensive studies during the last 5 years, we know thatseveral pathogenic mycoplasmas can rapidly alter the expres-sion and structure of their major surface proteins (reviewed inreference 35), a strategy which may contribute to their successas causative agents of chronic diseases through avoidance ofdefense mechanisms or adaptation to specialized environmentswithin their respective hosts. The ranges of mycoplasma spe-cies, structural motifs, and genetic systems involved in thissurface variability, however, are not fully defined. In this studywe have characterized a newly discovered system of myco-plasma surface antigenic variation in the bovine pathogen M.bovis. This system includes three variable surface membraneproteins, VspA, VspB, and VspC, which are antigenically andstructurally related members of a protein family.The distinction among three translational products was

based on their MAb and PAb epitope profiles and on theirpatterns of degradation at carboxypeptidase Y pause sites.However, while these structural fingerprints could easily dis-tinguish between VspA and VspB, as well as between VspCand VspB, the digestion patterns of VspA and VspC wereremarkably similar (Fig. lc and d). In the first place, thisobservation suggests that VspA and VspC might represent sizevariants of the same protein, differing only in a deletion of anN-terminal region. However, although this model would ex-plain the apparent 10-kDa size difference of the intact VspAand VspC by SDS-PAGE and would also predict a similarenzymatic digestion profile with the whole digestion pattern ofVspA shifted downwards, it is not consistent with the findingthat VspA and VspC can be coexpressed and are oscillating inexpression independently of one another (data not shown).From these results, we concluded that VspA and VspC shouldbe considered as separate entities.

Several characteristics of the Vsp protein family closelyresemble the Vlp prototype system ofM hyorhinis (5, 23-25,32, 35, 37). These include (i) association with lipid, (ii)membrane anchorage by N-terminal domains, (iii) regions ofshared epitopes, (iv) a surface-exposed C-terminal region withextensive repetitive structure, (v) high-frequency size variation,and (vi) high-frequency phase variation and combinatorialexpression. Moreover, both the Vlps ofM hyorhinis and theVsps of M bovis were shown to be the most abundantamphiphilic proteins in these organisms, as judged by therelative intensities of either their autoradiographic signals aftermetabolic labeling with [35S]cysteine (Vsps in Fig. 2c; Vlps inFig. lb of reference 25) or [3H]palmitate (Vsps in Fig. 2b; Vlpsin Fig. ld, of reference 25) or their staining in immunoblots(Vsps in Fig. 2a; VlpC in Fig. 2A, lane 4, of reference 24). Inaddition, both systems represent the major antigens on themembrane surface, which are predominantly responsible forthe host antibody response during natural or experimentalMbovis (22) and M hyorhinis (25) infections, respectively. Thehighly immunogenic nature of these antigens could serve to aidthe organisms in diverting and evading the host immunesystem, since it has been shown that surface proteins contain-ing immunodominant epitopes may influence the host antibodyresponse, resulting in interference with or subversion of thatresponse to other important molecules (13). Moreover, phasevariation in Vsp and Vlp expression may ensure survival ofthese pathogens, even in the presence of cytolytic antibodies(25).

Despite these overall similarities among the Vsp and Vlpsystems, there are, however, some key differences regardingthe detailed structure and size variations of the Vsp and Vlpmolecules. Although the precise molecular structures of theVsp antigens and the molecular basis of Vsp size variation arenot yet established, comparison of carboxypeptidase Y-gener-ated Vsp (Fig. 4) and Vlp (Fig. 4B in reference 24) ladderpatterns shows striking differences between the C-terminalperiodic structures of the Vsp and Vlp molecules. While thehighly regular Vlp ladder pattern has indicated the presence oftandemly repeated identical or nearly identical structural units(24, 25) (a conclusion later confirmed by DNA sequenceanalysis [37]), the nonequidistant Vsp ladder pattern stronglyargues for the occurrence of multiple sets of similar ornonsimilar repeat blocks, a molecular motif which might bequite analogous to that found in the size-variant M-proteinsurface antigens of group A streptococci (8, 10). It is alsointriguing and noteworthy that partial cleavage of Vsp variantswith trypsin generated only one (VspB) or two (VspA andVspC) immunodetectable fragments (Fig. 5), while trypsintreatment of Vlp variants yielded ladders similar to thoseobtained with carboxypeptidase Y (Fig. 4A in reference 24).This unexpected result predicts that the trypsin cleavage sitesand the repetitive pause sites defined by carboxypeptidase Ytruncation of Vsps are provided by nonidentical amino acidresidues, whereas within the Vlp molecules both sites arerepresented by Lys residues (37). Perhaps the most intriguingand novel feature of the Vsp system is its ability to generatesize variants not only by structural changes within the C-terminal repetitive structure analogous to those reported forthe Vlp system (24, 25, 37) but also by similar changes withinother regions of the Vsp molecules (Fig. 4 and 5), indicatingthat repetitive intragenic sequences which may be subjected toprecise deletion or insertion either by homologous recombina-tion (1) or by error-prone replicative processes (15) occur infact in various locations within the Vsp coding regions.These differences emphasize the uniqueness of the Vsp

family, and sequence analysis of the corresponding vsp geneswhich have been recently cloned (21, 36) will ultimatelyprovide answers to important questions involving not only themolecular structures of Vsp translation products but also themolecular mechanisms underlying Vsp size and phase varia-tion. Since the Vsp family of M. bovis provides a rapidlychanging surface mosaic that may profoundly affect interac-tions with its host environment and may therefore also accountfor differences in pathogenic potential among strains, anunderstanding of the molecular basis and regulation of thissurface variation will be critical in understanding the patho-genesis and immunology of M. bovis infections and mayprovide future opportunities to prevent them.

ACKNOWLEDGMENTSWe thank M. Ahrens and R. Schmidt for preparing the antiserum

used in this study.This work was supported by grant Ro 739/2-1 to R.R. from the

Deutsche Forschungsgemeinschaft.

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