INFECTION AND IMMUNITY, Nov. 1982, p. 668-6720019-9567/82/110668-05$02.00/0

Vol. 38, No. 2

Antigenicity of Neisseria gonorrhoeae Outer MembraneProtein(s) III Detected by Immunoprecipitation and Western

Blot Transfer with a Monoclonal AntibodyJOHN SWANSON,'* LEONARD W. MAYER,1 AND MILTON R. TAM2

Department of Health and Human Services, Public Health Service, National Institutes of Health, NationalInstitute ofAllergy and Infectious Diseases, Laboratory ofMicrobial Structure and Function, Rocky

Mountain Laboratories, Hamilton, Montana 59840-29991; and Genetic Systems, Seattle, Washington 981212

Received 26 April 1982/Accepted 29 July 1982

Some properties of gonococcal outer membrane III were studied by using a

monoclonal antibody (2E6) in Western blot transfer and immunoprecipitationreactions. By Western blot, the 2E6 monocAbnal had a single antigenic target:outer membrane protein III. All three gonococcal strains examined exhibitedidentical reactivities for their protein(s) III with this monoclonal. When 1251_labeled gonococci were incubated with monoclonal antibody 2E6, lysed inZwittergent, and antibody-antigen complexes were immunoprecipitated withprotein A-Sepharose, both outer membrane proteins I and III were present in theimmunoprecipitates regardless of the gonococcal strain used. Exposure of gono-cocci to Iodogen and 2-mercaptoethanol influences the electrophoretic migrationcharacteristics of protein(s) III in polyacrylamide gel electrophoresis with sodiumdodecyl sulfate; the antigenicity of protein(s) III was unaltered when assessed byWestern blot transfer after reaction with Iodogen or 2-mercaptoethanol. Thesedata demonstrate that outer membrane protein III is a surface-exposed antigenicmoiety common to diverse strains of gonococci.

Interactions of gonococci with their outsideworld occur via the surface components of theorganisms, so definition and characterization ofgonococcal surface constituents might yieldclues about the "personalities" and behavioralcapabilities of these pathogenic bacteria. Mostrecent studies have focused on two sets orfamilies of gonococcal outer membrane proteins(1-4, 9, 10, 12-14); these are protein I (principalor major outer membrane protein) and protein II(heat-modifiable or opacity-associated proteins).When outer membrane proteins are resolved inpolyacrylamide gels and visualized by Coomas-sie blue staining or autoradiography of lactoper-oxidase plus peroxide radioiodinated prepara-tions, the protein I and protein II moietiespredominate (2-4, 13). Neither of those methodsallows easy or striking definition of outer mem-brane protein(s) III; however, protein(s) III labelheavily and are easily visualized after Iodogen-catalyzed radioiodination and autoradiography(1, 15). Protein III (or protein 3, 3*) exhibitsretarded electrophoretic migration when solubi-lized in 2-mercaptoethanol (5, 6, 9). In situ, thisouter membrane protein resists proteolysis, incontrast to all protein II and many protein Ispecies (13, 16). Several different kinds of evi-dence suggest that protein III and protein I formheteropolymers on the exterior of the gonococ-

cus (9, 10). Judd used 125I-peptide mapping tostudy comparative structures of the 30,000- and31,000-dalton apparent subunit forms of pro-tein(s) III from four different strains (5, 6). Hefound that both overall and surface-labeled 1251_peptides of protein(s) III were virtually identi-cal. Although these outer membrane proteins IIIcan be radioiodinated on intact gonococci andseem identical by 125I-peptide mapping, do theypresent similar or identical antigenic moieties onthe exterior of the gonococcus? We have ap-proached that question by immunoprecipitationand Western blot methods with a monoclonalantibody directed against protein III.

MATERIALS AND METHODSGonococci. Strains JS1, JS3, and JS5 were used for

immunoprecipitation or Western blot transfer afterpropagation on a clear medium (12) in 5% CO2 at 36°Cfor 19 to 21 h. Gonococci of desired piliation, colonialopacity, and protein II phenotypes were obtained bysingle-colony serial passage. The particular strainsused were chosen because they differ in apparentmolecular weights of their protein I subunits.Monoclonal antibody production. Monoclonal

immunoglobulin G (IgG) antibodies were obtainedfrom fusions of BALB/c NSI/1 cells with lymph nodeand spleen cells from BALB/c mice immunized withpartially purified gonococcal outer membrane compo-nents. The particular IgG monoclonal 2E6 used here

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GC OUTER MEMBRANE PROTEIN III ANTIGENICITY 669

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FIG. 1. Western blot transfer with monoclonal 2E6and whole gonococcal lysates from three strains.Whole cell lysates from P+II- gonococci of strains JS1(1), JS3 (3), and JS5 (5) were resolved by SDS-PAGE,and the constituents were transferred to nitrocellulosewhich was incubated sequentially in monoclonal 2E6(1:500 dilution) or normal mouse serum (NMS) (1:500dilution) and 125I-labeled protein A. Radioiodinatedgonococci ([125 ]GC) of the same strains and pheno-types were included in the gel and were transferred tonitrocellulose; their autoradiograms were used forreference. The protein I subunits (*) for these threestrains differ (JS1 = 36,000 daltons, JS3 = 35,400daltons, and JS5 = 36,000 daltons), but their protein(s)III moieties are identical in electrophoretic character-istics. Monoclonal 2E6 antibody plus 1251I-labeled pro-tein A binds only to the protein(s) III bands, and allthree strains exhibit this reactivity. In the presence of2-mercaptoethanol (+2-ME), the proteins III haveapparent subunit molecular weights of 31,000; in theabsence of 2-mercaptoethanol (-2-ME), proteins IIIappear to be 30,000 daltons by comparison with Bio-Rad molecular-weight markers. Proteins III react withthe 2E6 monoclonal after electrophoresis both in thepresence (+2-ME) and the absence (-2-ME) of 2-mercaptoethanol. No reactivity of GC constituentswith NMS is visible at this serum dilution and with theautoradiographic exposure conditions used. At lowerdilutions, reactivities of NMS antibodies with proteinsI and III can be visualized (data not shown).

resulted from immunization of mice with a mixture ofthree partially purified protein I preparations de-scribed by Buchanan and Hildebrandt (2) from gono-coccal strains F62, 7122, and 8660. Detection of anti-body production by the fused cells, characterization ofthe immunoglobulin isotype, and production of ascitesfluids containing antibody-producing hybrid cells havebeen described in detail elsewhere (17).

Immunoprecipitation. Whole gonococci were radio-iodinated with 1,3,4,6-tetrachloro-3a, 6a-diphenylgly-coluril (lodogen, Pierce Chemical Co., Rockford, Ill.)(15) and incubated with either monoclonal antibody orBALB/c serum (1:6 final dilutions). After washing, theorganisms were lysed in a dipolar ionic detergent (N-tetradecyl -N, N-dimethyl-3-ammonio-1-propanesul-fonate; Zwittergent 3-14; Calbiochem-Behring Corp.,La Jolla, Calif.), and IgG plus 125I-labeled gonococcal

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-2-MEFIG. 2. Influence of lodogen and 2-mercaptoeth-

anol on migration characteristics of protein(s) III de-tected by monoclonal 2E6 in Western blot transfer.Whole gonococci of strain JS3 were exposed to lodo-gen (IDGN) with or without 125I (Na 1251) and potassi-um (KI) iodide before solubilization for SDS-PAGEwith (+2-ME) or without 2-mercaptoethanol (-2-ME).After SDS-PAGE, electrophoretic transfer of gono-coccal components to nitrocellulose was followed byincubation in monoclonal 2E6 and 125I-labeled proteinA to localize protein(s) III; the resultant autoradio-gram is shown. All specimens exposed to lodogenexhibit slightly slowed migration of protein(s) III ascompared with those not exposed to this iodinatingreagent; this is seen both in the presence (+2-ME) andabsence (-2-ME) of 2-mercaptoethanol. In the pres-ence of 2-mercaptoethanol, lodogen-treated proteinIII appears as two bands (:). Protein III appears as asingle band (o) in the absence of lodogen treatment(with or without 2-mercaptoethanol) and in the lodo-gen-treated specimens solubilized in the absence of 2-mercaptoethanol.

components were recovered by incubating the deter-gent lysate with protein A-Sepharose 4B CL (SigmaChemical Co., St. Louis, Mo.). Radioiodinated com-ponents in the immunoprecipitates were resolved bypolyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate (SDS-PAGE) and were dis-played by autoradiography. This procedure has beendescribed in detail elsewhere (15).

Electrophoretic transfer to nitroceliulose and anti-body localization. Gonococci (1.5 ml; optical density at540 nm = 0.6; pelleted and diluted in 100 ,ul ofphosphate-buffered saline) were suspended in phos-phate-buffered saline; after lysis of 2 ,u of gonococcalsuspension in 8 ,ul of SDS-PAGE solubilizing solution(4% SDS, 1.25 mM Tris-hydrochloride, 20% glycerol,pH 6.8, with or without 8% 2-mercaptoethanol) in aboiling water bath for 10 min, the whole cell lysateswere subjected to SDS-PAGE in 12.5% gels in thebuffer system described by Laemmli (7). Radioiodi-nated organisms of the same strains and phenotypeswere also included in each slab gel for identification ofgonococci proteins. At the end of SDS-PAGE, the slabgel was apposed to a sheet of nitrocellulose (MilliporeHAHY, Millipore Corp., San Francisco, Calif.) towhich proteins were electrophoretically transferred inthe methanol-Tris-glycine system described by Tow-bin et al. (18). After electrophoresis (3 h, 60 V, in Bio-Rad Trans-Blot apparatus, Bio-Rad Laboratories,Richmond, Calif.), the nitrocellulose sheet was

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FIG. 3. Immunoprecipitation of 125I-labeled gono-coccal proteins after exposure of protein II- gono-cocci to monoclonal 2E6. Gonococci with P'Il- phe-notypes from strains JS1 (1), JS3 (3), and JS5 (5) wereradioiodinated and incubated with monoclonal anti-body 2E6 (1:500) or with similarly diluted normalmouse serum (NMS). This was followed by lysis inZwittergent 3-14 and recovery of antibody-antigencomplexes with protein A-Sepharose 4B CL. In the1251I-labeled gonococcal transfer controls ([125I]GC),proteins I and III are clearly visualized. Both proteinsI and III are found in the immunoprecipitates obtainedwith monoclonal 2E6, whereas no radioiodinated com-ponents are found after treatment with normal mouseserum (NMS). Note the apparent "splitting" of pro-tein III into two bands (=#) in the immunoprecipitates.Note also the different apparent subunit sizes forprotein(s) I for these three strains (*) in the [125I]GC;these differences are clearly visible in the immunopre-cipitates with 2E6.

washed, soaked overnight in 2% bovine serum albu-min (Pentax Fraction V, Miles Laboratories, Inc.,Elkhart, Ind.), and incubated for 2 h with monoclonalantibody or BALB/c mouse serum (each diluted1:500); albumin, monoclonal antibody, and mouseserum were diluted in the Tris-NaCl-gelatin-NonidetP-40 solution described by Renart et al. (11). Afteradditional washing, the sheets were incubated withprotein A (Sigma) that was radioiodinated by andobtained from Alan Barbour (Laboratory of MicrobialStructure and Function). Specific activity of the 1251_labeled protein A was approximately 5 uCi/,Lg. Local-ization of the radiolabeled protein A was accomplishedwith X-ray film (Kodak XAR) exposed to the washedand dried nitrocellulose sheet for 24 h at room tem-perature. This technique for electrophoretic transfer tonitrocellulose sheets and their incubation with anti-body and 125I-labeled protein, etc., will be referred toas Western blot transfer.

RESULTS

Monoclonal 2E6 reactivity by Western blottransfer. Whole gonococci from three strainswere subjected to SDS-PAGE, and their pro-teins were transferred to nitrocellulose for incu-bation with the monoclonal 2E6 or normal

FIG. 4. Immunoprecipitation of "251-labeled gono-coccal proteins after exposure of protein II+ gono-cocci to monoclonal 2E6. Gonococci of strain JS3 andJS1 that bear three and two protein II moieties,respectively, were radioiodinated and incubated witheither monoclonal 2E6 or another ascites fluid mono-clonal (2F8), each diluted 1:500. Proteins I and III areimmunoprecipitated by the 2E6. Protein II moietiesare absent in the immunoprecipitates, nor does theirpresence interfere with reaction of the 2E6 monoclonalantibodies with proteins I and III (compare with Fig.3). Another ascites fluid monoclonal antibody prepara-tion (2F8) displayed no reactivity against proteins I,III, or III and is included as a control. The protein IIconstituents present on gonococci in this experimentare indicated as follows and defined in an earlier study(16): JS3 P.Ia (0); P.IIb (O); P.Ie (0); JS1 P.11a (-);and P.IIP (>).

mouse serum (Fig. 1). The 2E6 monoclonalreacted only with protein III in the three strainsexamined. Both the slow- (presence of 2-mer-captoethanol) and fast- (absence of 2-mercap-toethanol) migrating forms of protein(s) III werevisualized by autoradiographic localization of125I-protein A that binds to the IgG moieties ofthe monoclonal. Although difficult to see in Fig.1, the monoclonal 2E6 antibody and 125I-labeledprotein A localized in bands that were slightlylower in the gel than the protein III seen in thecompanion transfer controls of 125I-labeled gon-ococci when both were solubilized in 2-mercap-toethanol (+2-ME). This subtle reduction inmigration occurred in the presence and absenceof 2-mercaptoethanol for protein(s) III aftertheir exposure to Iodogen (Fig. 2). Exposure tolodogen also resulted in migration of protein(s)III as two discrete bands in the presence of 2-mercaptoethanol; this band splitting was onlyobvious when 2-mercaptoethanol was presentduring solubilizing for SDS-PAGE. Both proteinIII bands bound monoclonal 2E6 antibody.

Immunoprecipitation. Gonococci that lackedprotein II components were prepared from thesame three strains described above and wereused for immunoprecipitation of their surface-exposed, 125I-labeled components by the mono-clonal antibody preparation. Both protein I and

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GC OUTER MEMBRANE PROTEIN III ANTIGENICITY 671

protein III appeared in the immunoprecipitatesfrom all three strains (Fig. 3). In these prepara-tions, protein III appeared to have split into twocomponents; this was a characteristic of proteinIII moieties exposed to the lodogen reagentduring radioiodination, as noted above. No im-munoprecipitation of 125I-labeled constituentswas seen for gonococci incubated with normalmouse serum at the dilutions used.For two strains, opaque-colony gonococci

having protein II constituents were used forimmunoprecipitation. These enhanced-opacity,protein II' variants were used to determinewhether protein III constituents were "visible"on the surfaces of gonococci that also had madeheat-modifiable outer membrane protein II moi-eties. The immunoprecipitation results areshown in Fig. 4. Both proteins I and III wereimmunoprecipitated, but no protein II.

DISCUSSIONThe results described above make several

points regarding outer membrane protein(s) IIIof gonococci, as follows.

(i) Both "native" and SDS-denatured formsof protein(s) III contain an antigenic epitoperecognized by the monoclonal antibody 2E6;this antigenicity is retained after lodogen or 2-mercaptoethanol treatment of protein(s) III as-sessed by Western blot transfer.

(ii) Proteins III are "visible" on the exteriorsof gonococci, as shown by the ability of the anti-protein III monoclonal 2E6 to immunoprecipi-tate protein III after incubation with intact, 125I-labeled gonococci.

(iii) The protein(s) III for the three gonococ-cal strains examined had identical reactivitieswith the monoclonal 2E6 by both immunopre-cipitation and Western blot transfer analyses;thus, protein III is "surface-exposed" antigencommon to diverse (if not all) gonococcalstrains.

(iv) Proteins I and III exist as complexes inthe detergent Zwittergent, but these complexesare dissociated by SDS. These phenomena leadto "coprecipitation" of protein I during immun-oprecipitation of protein III with anti-protein IIImonoclonal antibody. Protein I plus protein IIIcomplexes are found regardless of the apparentsubunit molecular weight of the gonococcal out-er membrane protein I.The immunochemical characteristics of pro-

tein(s) III have not been clearly defined inprevious studies that utilized antisera from rab-bits immunized with whole gonococci (15), "se-rotype-specific vesicles" (9), or "purified pro-tein I" constituents (2). When either wholeorganisms or outer membrane vesicles are usedboth as immunogens and antigens, both protein Iand protein(s) III are immunoprecipitated (9,

15). Because proteins I and III occur in immuno-precipitates in similar ratios, results with ho-mologous and heterologous strains and antiserasuggested that antibodies were directed, primar-ily, against protein I constituents and that pro-teins III were "hitchhikers" by virtue of theirclose association with protein I subunits. Aprevious study utilized Western blot transferand antisera raised against "purified protein I;"it shows apparent identical or similar antigenicreactivities of protein(s) III of homologous andheterologous strains (Fig. 5 of ref. 2), but thosefindings are not mentioned by the authors. In thepresent study, proteins III were recognized by amonoclonal antibody by Western blot transfer toclearly establish that gonococcal outer mem-brane protein III per se is indeed antigenic. Notonly is protein III antigenic, but it appears to bea surface-exposed antigen present in diversegonococcal strains. Such surface exposure issuggested by immunoprecipitation experimentssince the 125I-labeled bacteria were incubatedwith the anti-protein III monoclonal antibodybefore lysis of the organisms in detergent. Thepresence of both protein(s) I and III in theresultant immunoprecipitate adds additional evi-dence for interactions between these two outermembrane proteins in the presence of selecteddetergents such as Zwittergent; this protein I-protein III interaction is disrupted by boiling inSDS. These phenomena support previous obser-vations which suggested that proteins I and IIIform "hydrophobically associated trimeric unitin situ which can be stabilized by selectivecross-linking reagents" (9, 10).

Alterations in the electrophoretic migrationcharacteristics of protein(s) III after exposure toIodogen may reflect cleavage of sulfhydrylgroups, as was reported by McClard (8). We donot understand the appearance of doublet bandsfor protein(s) III after lodogen treatment andelectrophoresis with 2-mercaptoethanol. Modifi-cation of protein(s) III by Iodogen or 2-mercap-toethanol or both does not appear to compro-mise the antigenicity of this outer membraneconstituent.Although the present study does not define a

contribution by protein(s) III in serotypic reac-tions, it is clear that this outer membrane proteinis exposed on the exterior of the organism and isprobably present in many, if not all, gonococcalstrains. Further, the antigenicities of protein(s)III assessed by either immunoprecipitation orWestern blotting techniques are not changed bythe coexistence of protein I constituents of di-verse subunit sizes and 125I-labeled peptide pat-terns.

LITERATURE CITED1. Blake, M. S., E. C. Gotschlich, and J. Swanson. 1981.

Effects of proteolytic enzymes on the outer membrane

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proteins of Neisseria gonorrhoeae. Infect. Immun. 33:212-222.

2. Buchanan, T. M., and J. F. Hildebrandt. 1981. Antigen-specific serotyping of Neisseria gonorrhoeae: character-ization based on principal outer membrane protein. Infect.Immun. 32:985-994.

3. Heckels, J. E. 1977. The surface properties of Neisseriagonorrhoeae: isolation of the major components of theouter membrane. J. Gen. Microbiol. 99:333-341.

4. Heckels, J. E. 1981. Structural composition of Neisseriagonorrhoeae outer membrane proteins. J. Bacteriol.145:736-742.

5. Judd, R. C. 125I-peptide mapping of protein III isolatedfrom four strains of Neisseria gonorrhoeae. Infect. Im-mun. 37:622-631.

6. Judd, R. C. Surface peptide mapping of protein I andprotein III of four strains of Neisseria gonorrhoeae.Infect. Immun. 37:632-641.

7. Laemmnl, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

8. McClard, R. W. 1981. Removal of sulfhydryl groups with1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril: applicationto the assay of protein in the presence of thiol reagents.Anal. Biochem. 112:278-281.

9. McDade, R. L., Jr., and K. H. Johnston. 1980. Character-ization of serologically dominant outer membrane pro-teins of Neisseria gonorrhoeae. J. Bacteriol. 141:1183-1191.

10. Newhall, W. J., V, W. D. Sawyer, and R. A. Haak. 1980.

Cross-linking analysis of the outer membrane proteins ofNeisseria gonorrhoeae. Infect. Immun. 28:785-791.

11. Renart, J., J. Reiser, and G. R. Stark. 1979. Transfer ofproteins from gels to diazobenzyloxymethyl-paper anddetection with antisera: a method for studying antibodyspecificity and antigen structure. Proc. Natl. Acad. Sci.U.S.A. 76:3116-3120.

12. Swanson, J. 1978. Studies on gonococcus infection. XII.Colony color and opacity variants of gonococci. Infect.Immun. 19:320-331.

13. Swanson, J. 1979. Studies on gonococcus infection.XVIII. 1251-labeled peptide mapping of the major proteinof the gonococcal cell wall outer membrane. Infect.Immun. 23:799-810.

14. Swanson, J. 1980. 1251-labeled peptide mapping of someheat-modifiable proteins of the gonococcal outer mem-brane. Infect. Immun. 28:54-64.

15. Swanson, J. 1981. Surface-exposed protein antigens of thegonococcal outer membrane. Infect. Immun. 34:804-816.

16. Swanson, J. 1982. Colony opacity and protein II composi-tions of gonococci. Infect. Immun. 37:359-368.

17. Tam, M. R., T. M. Buchanan, E. G. Sandstr6m, K. K.Holmes, J. S. Knapp, A. W. Siadak, and R. C. Nowinski.1982. Serological classification of Neisseria gonorrhoeaewith monoclonal antibodies. Infect. Immun. 36:1042-1053.

18. Towbin, H., T. Staehelln, and J. Gordon. 1979. Electro-phoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: procedure and some applications.Proc. NatI. Acad. Sci. U.S.A. 76:4350-4354.

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