Vol. 37, No. 2INFECTION AND IMMUNITY, Aug. 1982, p. 632-6410019-9567/82/080632-10$02.00/0

Surface Peptide Mapping of Protein I and Protein III of FourStrains of Neisseria gonorrhoeae

RALPH C. JUDDLaboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute ofAllergy

and Infectious Diseases, Hamilton, Montana 59840

Received 21 December 1981/Accepted 9 April 1982

Whole cells and isolated outer membranes (OMs) of four strains of gonococciwere surface radioiodinated with either lactoperoxidase or lodogen (PierceChemical Co., Rockford, Ill.). These preparations were solubilized in sodiumdodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excisedfrom the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels anddigested with a-chymotrypsin, and the resultant 125I-peptide fragments wereresolved by high-voltage electrophoresis and thin-layer chromatography (i.e.,surface peptide mapping). Radioemitting peptidic fragments were visualized byautoradiography. Results demonstrated that the PI molecule of each gonococcalstrain studied had unique iodinatable peptides exposed on the surface of wholecells and OMs, whereas PIlls appeared to have the same portion of the moleculeexposed on the surface of bacteria or OMs, regardless of the gonococcal strainfrom which they were isolated. Many more radiolabeled peptides were seen insurface peptide maps of PIs from radiolabeled OMs than in those from radioiodi-nated whole cells, whereas different peptidic fragments were seen in the surfacepeptide maps of PIIIs from radiolabeled OMs than were seen in those fromradiolabeled whole cells. These data suggest that PI may contribute strain-specificantigenic determinants and Pll may contribute cross-reactive determinants andthat the surface exposure of PI and PIII is different in isolated OMs than in the OMof intact gonococci.

The principal outer membrane (OM) protein(protein I [PI]) of Neisseria gonorrhoeae isthought to exist both as a homopolymer and in aheteropolymer with the 2-mercaptoethanol(2ME)-modifiable PIII (13, 15). In a previous'25I-peptide mapping study, PIs of differing ap-parent subunit molecular weights (MWs), asdefined by sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE), couldbe placed into two primary structure homologygroups, one encompassing the higher apparent-subunit-MW PIs, the other the lower apparent-subunit-MW PIs (20). More recently, it wasshown that the chymotryptic 125I-peptide mapsof PIIIs were very similar when isolated fromfour strains of N. gonorrhoeae having differentapparent-subunit-MW PIs (9). Taken together,the above observations suggest that PIIIs ofsimilar or identical primary structures form het-eropolymers with different PIs.

Immunoprecipitation experiments with rabbitantiserum raised against whole cells (21) or OMvesicles (13) have shown that PI and PIII copre-cipitate, making it difficult to establish whetherPllls are antigenic per se or whether PIII iscoprecipitated by antibodies directed against PI.

Since PIlIs appear to be the same in all N.gonorrhoeae strains thus far studied, PIII maycontribute cross-reactive surface antigens.However, it is also possible that association withdifferent PIs could expose different portions ofthe PIII molecule to the bacterial surface, mak-ing strain-specific antigens available for reactionwith specific antibody.A non-immunological approach to investigate

the nature of PI and PIII surface exposure wassuggested by the work of Heckels (6), in whichPIIs, the heat-modifiable, colony opacity-associ-ated proteins (5, 7, 19), from surface-radioiodi-nated OM vesicles were separated by SDS-PAGE and subjected to peptide mapping(surface peptide mapping). Those peptides avail-able for surface radioiodination were visualizedby autoradiography. Comparison of these sur-face peptide maps with peptide maps of Pllswhich were separated by SDS-PAGE, excisedfrom the gel, exhaustively radioiodinated by thechloramine-T procedure (3, 4), and subjected to125I-peptide mapping (chloro-T-125I-peptidemapping) demonstrated that PIIs of similar pri-mary structures had different radioiodinatedpeptides exposed on the OM surface. Subse-

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quent work in which similar procedures wereused to investigate PII surface exposure onintact N. gonorrhoeae correlated the presenceof unique surface-exposed peptides of PlIs,which had overall similar chloro-T-125I-peptidemaps, with immunological specificity (J. Swan-son, 0. Barrera, and R. C. Judd, manuscript inpreparation).

In this study, surface peptide mapping wasused to determine which radioiodinatable pep-tides of PI and PIII are exposed on the exteriorof intact bacteria and OM vesicles from fourstrains of N. gonorrhoeae. Two reagents forcatalyzing radioiodination, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril (Iodogen; PierceChemical Co., Rockford, Ill.) and lactoperoxi-dase (LPO), were used to surface label wholecells. lodogen was also used to surface iodinateOM vesicles. The radiolabeled PIs and PIlIswere separated by SDS-PAGE and then subject-ed to peptide mapping. The resultant surfacepeytide maps were compared with chloro-T-

I-peptide maps of these proteins to establishthe nature of PI and PIII surface exposure.The results of these analyses demonstrated

that the PI molecule of each N. gonorrhoeaestrain studied had unique iodinatable peptidesexposed on the surfaces of whole cells or OMs,whereas PIlls appeared to have the same sur-face-exposed peptides. In addition, the surfacepeptide maps of PIs and PIlls were different inOMs than in whole cells, suggesting that thesemolecules have a different surface exposure inOMs than they do in whole cells.

MATERIALS AND METHODSBacteria. N. gonorrhoeae strains JS1 (original desig-

nation, F62), JS2 (original designation, 10677-3), JS3(original designation, 120176-3), and JS4 (original des-ignation, MSL-7040, 1972) were grown on clear typingmedium as previously described (18). Organisms weregrown at 36°C in 5% CO2 for 18 to 21 h. Nonpiliated(P-), transparent (O-) organisms were used through-out this study.OM preparations. OM vesicles were prepared by

shaking whole N. gonorrhoeae, freshly isolated byswabbing bacteria from agar plates, in 0.1 M Tris-1 MNaCl-0.02% sodium azide buffer (pH 8.0) at 43°C with0.3-mm-diameter glass beads. Vesicles were then iso-lated by differential centrifugation and column chro-matography (Sepharose 6B; Pharmacia Fine Chemi-cals, Inc., Piscataway, N.J.) (20) and stored at -20°Cuntil used.

Radloiodination of whole bacteria. Two techniqueswere used to surface radioiodinate whole N. gonor-rhoeae, the LPO procedure (16, 19) and the lodogenreaction (11).LPO labeling. N. gonorrhoeae cells were swabbed

from agar plates and suspended in Dulbecco phos-phate-buffered saline (DPBS), pH 7.4, to a turbidity of200 Klett units (blue filter). A 1.5-ml amount of thissuspension was centrifuged in a Microfuge B (Beck-

man Instruments, Inc., Fullerton, Calif.), and theresultant bacterial pellet was washed three times inDPBS. The final pellet was resuspended in 25 ,ul ofDPBS containing 0.8 mg of LPO (Sigma Chemical Co.,St. Louis, Mo.) per ml. To this was added 5 ,u of 10-5M KI and 4 ,ul of 125I (as Nal; 50 ,uCi/,ul). At 0, 2.5, 5,7.5, and 10 min, 5 ,ul offresh 0.3% H202 was added. At12.5 min, the bacteria were pelleted by centrifugationand washed three times in DPBS with 0.005 M cys-teine. The radioiodinated bacteria were resuspendedin 500 p.l of DPBS. A 50-Rl amount of this suspensionwas added to 50 pll of the appropriate SDS solubilizingsolution, and S pul of this lysate was subjected to SDS-PAGE (see below).

Iodogen labeling. Bacteria were harvested andwashed as described in the LPO procedure. To thewashed bacterial pellet was added 42 ,ul of DPBS and 4pul of 10-5 M KI. The bacterial suspension was addedto an Iodogen-coated microfuge tube (prepared byadding 10 p.g of lodogen to 10 ,ul of chloroform andthen allowing the chloroform to evaporate). A total of4 pul of 125I (as NaI; 50 ,uCi/,ul) was added, and the tubewas left on ice for 10 min. After this brief incubation,the reaction was terminated by aspirating the bacterialsuspension. The radioiodinated N. gonorrhoeae cellswere washed three times in DPBS, divided into ali-quots, and solubilized as described in the LPO proce-dure.

Iodination ofOM vesicles. Isolated OM vesicles wereradioiodinated by the lodogen procedure. To an lodo-gen-coated Microfuge tube (as described above) wasadded 42 pul of the appropriate OM preparation and 4,ul of 10-5 M KI. A total of 4 Ill of 1251 (as NaI; 50 ,uCi/p.1) was then added, and the reaction was allowed toproceed for 10 min on ice. The reaction was halted byaspirating the OM suspension. The aspirate was divid-ed into two aliquots. One aliquot was immediatelysolubilized in an equal volume of SDS solubilizingsolution (see below) without 2ME, and the otheraliquot was SDS solubilized with 2ME. After solubili-zation, these preparations were subjected to SDS-PAGE.SDS-PAGE. Proteins to be used in peptide mapping

were separated on 15% acrylamide (acrylamide/N,N'-methylenebisacrylamide ratio, 30:0.8) slab gels by theTris-glycine system of Laemmli (10). Whole cells orOMs were solubilized at 100°C for 5 min in 10%o (wt/vol) SDS-10%o (vol/vol) glycerol-0.1 M Tris (pH 6.8)solubilizing solution either with or without 8% 2ME.Samples were electrophoresed, fixed, stained, dried,and autoradiographed as previously described (21).

'2sI-peptide mapping. The radioiodination of proteinbands excised from SDS-PAGE gels with chloramine-T, proteolysis with a-chymotrypsin, high-voltage thin-layer electrophoresis, ascending thin-layer chromatog-raphy, and autoradiography, referred to as chloro-T-125I-peptide mapping, were performed as previouslydescribed (3, 9, 20).

Surface peptide mapping. PI and PIII Coomassiebrilliant blue-stained bands from surface-radioiodi-nated whole cells and OMs of each N. gonorrhoeaestrain were excised from SDS-PAGE gels. The gelslices were soaked overnight in 15% methanol andthen dried in a Speed-Vac (Savant Instruments, Inc.,Hicksville, N.Y.). The gel slices were then rehydratedin 250 p.l of 0.05 M ammonium bicarbonate (pH 8.5). A

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FIG. 1. Autoradiograms oiof whole-cell (WC) and OMgonorrhoeae strains JS1, JS2,cells (unreduced) radioiodinEthen SDS solubilized withou(reduced) radioiodinated withsolubilized in the presence of(reduced) radioiodinated witsolubilized in the presence ofradioiodinated OMs (unrediwithout 2ME; (E) Iodogen-rduced) SDS solubilized in thePIs; III, 30K PIIIs (unreduc31K PIIls (reduced form ofdoublet 31K PII1*s (reduce(occurred when whole cells orwith the lodogen reagent andthe presence of 8% 2ME. Orgels are shown.

25-,ul amount ofa-chymotrypsCorp., La Jolla, Calif.; 1 mgadded, and digestion was allov37°C. The supematant was asdigestion process was repeateue for 16 h at 37°C. The saspirated and combined with tthe preparation was dried on aic residue was then washeddistilled water. The washedsuspended in a minimum volusubjected to high-voltage thiascending thin-layer chromatoraphy, henceforth referred toping, as previously described

RESUL'SDS-PAGE of surface-

cells and OM vesicles. Iodused to surface radioiodinastrains of N. gonorrhoeaebacteria were then SDS soabsence or the presence oto SDS-PAGE. Autoradi(gels are shown in Fig. 1.

were heavily labeled with 125I by both Iodogen(Fig. IA and B) and LPO (Fig. 1C). PIIIs were

;t*. weakly iodinated by LPO, whereas lodogen't_~ radioiodinated the PIIIs at a level almost equiva-

lent to that of the PIs. The lodogen procedurecaused the PIII bands in reduced whole-celllysates to appear as two sharp bands (apparentsubunit molecular weight of 31,000 [31K] PIII*aand 31K PIII*b [Fig. 1B]), whereas LPO causeda smearing of the PIll bands in reduced whole-cell lysates (Fig. 1C). The doublet PIII band orsmearing of the PIII bands or both was not seenin unreduced whole-cell lysates (Fig. 1A), norwas it observed in either reduced or unreducedlysates of non-iodinated whole cells (9), indicat-ing that the oxidative iodination process, fol-lowed by SDS solubilization in the presence of

f ldred SDS-PAGE gels 8% 2ME, results in two populations of 31K PIlIsJS3, and JS4. (A) Whole having perhaps slightly different structures. Toated with lodogen and avoid confusion, only PIs and PIlls from unre-I 2ME; (B) whole cells duced whole-cell lysates were subjected to sur-Iodogen, and then SDS face peptide mapping.8% 2ME; (C) whole cells OM vesicles from each N. gonorrhoeae strainh LPO and then SDS were surface radioiodinated with the lodogen'8% 2ME; (D) Iodogen- reagent. These preparations were then solubi-uced) SDS solubilized lized in SDS in either the absence or the pres-adioiodinated OMs (re- ence of 2ME and subjected to SDS-PAGE. An

ed form of PIlls); III autoradiography of this dried gel is shown in Fig.Pilfs); III*a and III*b 1D and E. As with whole cells, both PIs andI form of PIII) which PIIls of all four strains were radioiodinated byOMs were radiolabeled this procedure. Again, the doublet PIII was seenthen SDS solubilized in in reduced OM lysates. The unreduced PInly relevant portions of bands, the unreduced PIII bands (30K PIII), and

both PIII bands seen in reduced OM lysates(31K PIII*a and 31K PIII*b) were excised andsubjected to surface peptide mapping.

sin (Calbiochem-Behring Surface peptide mapping of PIs from lysates of/ml in 0.01 N HCI) was radioiodinated whole cells. PI bands from SDS-ved to continue for 4 hat PAGE of lysates from Iodogen-radioiodinatedpirated and frozen. The and LPO-radioiodinated whole cells of the fourd but allowed to contin- N. gonorrhoeae strains under study were ex-;econd supematant was cised and subjected to surface peptide mapping.the first supernatant, and Autoradiographs of these preparations areSpeed-Vac. The peptid- soni i.2four times in 250 1d1 of shown in Fig. 2.peptidic residue was re- Comparison of the Iodogen-labeled PI surfaceie of distilled H20 and peptide maps (Fig. 2A) with the LPO-radioiodi-in-layer electrophoresis, nated PI surface peptide maps (Fig. 2B) showed)graphy, and autoradiog- that both procedures qualitatively and quantita-as surface peptide map- tively radiolabeled the same peptides in each PI.(3, 9, 20). The PIs of JS1 and JS2, which had similar

chloro-T-125I-peptide maps (Fig. 3B), also hadTS somewhat similar surface peptide maps (Fig. 2Aradioiodinated whole and B). There were, however, only a few com-logen and LPO were mon surface-exposed peptides in the JS1 andate whole cells of four JS2 PIs; most of the peptides were unique to onee. The radioiodinated or the other PI. The JS3 and JS4 PIs, which had)lubilized in either the similar chloro-T-125I-peptide maps, also had)f 2ME and subjected somewhat similar surface peptide maps, with)graphs of the dried some common peptides and several unique ra-The PIs of all strains dioemitting peptides.

634 JUDD

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FIG. 2. a-Chymotryptic surface peptide maps of PI bands excised from SDS-PAGE gels of unreduced lysatesof P- 0- whole cells (WCs) of N. gonorrhoeae strains JS1, JS2, JS3, and JS4. Whole cells were radiolabeledwith either lodogen (A) or LPO (B). Circles indicate radioemitting peptides that are not visualized in chloro-T-125I-peptide maps of these proteins (see Fig. 3B). TLE, Thin-layer electrophoresis; TLC, ascending thin-layerchromatography.

Those portions of the JS1 and JS2 PIs whichwere exposed to the bacterial surface had morea-chymotryptic-sensitive sites, as evidenced bythe number of peptides seen in these maps, thandid the surface-exposed portions of JS3 and JS4PIs. Extended exposure periods of up to 2 weeksrevealed no weakly emitting peptides not seen inthese figures for any of the PIs.

Surface peptide mapping of PIs from lysates ofIodogen-radioiodinated OM vesicles. OM vesi-cles were isolated, and the nature of the surface-radioiodinatable peptides was explored by sur-face peptide mapping. Autoradiographs ofsurface peptide maps of PIs from the four N.gonorrhoeae strains under study are shown inFig. 3A. As with the surface peptide maps of PIsfrom lodogen-surface-labeled whole cells, theJS1 and JS2 PI surface peptide maps from iodin-ated OM vesicles were similar to each other,whereas the surface peptide maps ofJS3 and JS4

PIs from surface-iodinated OMs were similar toone another.There were many more radioemitting peptides

in all of the surface peptide maps of PIs fromIodogen-radioiodinated OMs than there were inthose from lodogen-radiolabeled whole cells.Virtually all of the peptides seen in the chloro-T-125I-peptide maps of PIs could be visualized inthe surface peptide maps of PIs from Iodogen-radiolabeled OMs with extended exposure peri-ods (data not shown) but with very differentrelative intensities. Moreover, the PI surfacepeptide maps from Iodogen-radiolabeled OMshad a greater number of intensely emitting pep-tides than did those from Iodogen-radioiodi-nated whole cells. These observations suggestedthat the PI molecules may have different surfaceexposures in the membrane ofOM vesicles thanthey do in the OM of whole cells. Each OM PIhad several unique surface-exposed peptides.

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FIG. 3. (A) a-Chymotryptic surface peptide maps of PI bands excised from an SDS-PAGE gel of lysates ofOMs of P- 0- N. gonorrhoeae strains JS1, JS2, JS3, and JS4 which were radiolabeled with the Iodogen reagent.Circles indicate radioemitting peptides not visualized in chloro-T-125I-peptide maps of these proteins. (B) a-Chymotryptic chloro-T-1251-peptide maps of PI bands excised from an SDS-PAGE gel of lysates of whole cells ofP- 0- N. gonorrhoeae strains JS1, JS2, JS3, and JS4. This figure is included for comparison with the various PIsurface peptide maps. A, Peptides which appeared to correspond with radioemitting peptides seen in surfacepeptide maps of PI bands excised from an SDS-PAGE gel of lysates of radiolabeled whole cells. A, Peptideswhich appeared to correspond with radioemitting peptides seen in surface peptide maps of PI bands excised froman SDS-PAGE gel of lysates of radiolabeled OMs. TLE, Thin-layer electrophoresis; TLC, ascending thin-layerchromatography.

Interestingly, one of the chloro-T-'1251-peptidesthat was unique to the JS2 PI, as compared withJS1 PI (Fig. 3B), was a surface-exposed peptidein both the whole-cell and OM preparations.Those chloro-T-125I-peptides which seemed tobe unique to the JS3 PI, as compared with theJS4 PI, were seen in the surface peptide maps ofPIs from radiolabeled OM vesicles but not insurface peptide maps from whole cells.

Surface peptide mapping of PIIIs isolated fromlysates of radioiodinated whole cells. PIlIs wereexcised from SDS-PAGE gels of unreduced ly-sates from Iodogen-radioiodinated and LPO-radioiodinated whole cells and used for surfacepeptide mapping. Autoradiographs of these PIIIpreparations are shown in Fig. 4. Analogous to

the PI surface peptide mapping with whole cells,Iodogen and LPO appeared to label the samepeptides of Pllls to the same relative intensities.Slight differences in the positions of radioemit-ting peptides were felt to be due to technicalvariation. Unlike the PI surface peptide maps,the PIII surface peptide maps appeared to beidentical within the limits of this technique,suggesting that all Pllls have the same iodinata-ble peptides on the bacterial surface, regardlessof the PI type with which it is associated. Therewere fewer radioemitting peptides in the whole-cell PIII surface peptide maps than were seen inthe whole-cell PI surface peptide maps; thosepeptides which were seen appeared to be moreweakly iodinated than the peptides seen in the PI

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FIG. 4. a-Chymotryptic surface peptide maps of PIII bands excised from an SDS-PAGE gel of lysates ofwhole cells (WCs) ofP- 0- N. gonorrhoeae strains JS1, JS2, JS3, and JS4 which were radioiodinated with eitherlodogen (A) or LPO (B). TLE, Thin-layer electrophoresis; TLC, ascending thin-layer chromatography.

surface peptide maps, suggesting that less of thePIII molecule is available for surface iodinationand that those portions of the PIII moleculewhich are exposed have fewer reactive sites(i.e., tyrosine and histidine [14]). Extended ex-

posures did not reveal any weakly emittingpeptides not seen in these figures (data notshown).

Surface peptide mapping of PIIs isolated fromlysates of Iodogen-radioiodinated OM vesicles.PIII bands were excised from SDS-PAGE gelsof unreduced and reduced lysates of Iodogen-radiolabeled OM vesicles. Three PIII bandsfrom each strain were examined by surfacepeptide mapping. The 30K PIll band was takenfrom an SDS-PAGE gel of unreduced lysates ofradiolabeled OM vesicles. The 31K PIII*a and31K PIII*b bands (i.e., the doublet PIII* bandswhich resulted from Iodogen treatment) wereexcised from an SDS-PAGE gel of reducedlysates of radioiodinated OM vesicles. Autora-diographs of the surface peptide maps of these

bands are shown in Fig. 5. In each surfacepeptide map, the same intensely emitting pep-tides were seen. As with PIs from lodogen-labeled OM vesicles, extensive exposure re-vealed virtually all of the peptides seen in thechloro-T-125I-peptide maps of PIlIs (Fig. SD)but with different relative intensities. This wasnot seen in the surface peptide maps of PIllsfrom radioiodinated whole cells.Two of the surface-exposed peptides seen in

the surface peptide maps of PIIls from radioiodi-nated OM vesicles appeared to correlate withthose from radiolabeled whole cells. There weretwo other dominant peptides in surface peptidemaps of Pllls from labeled OM vesicles whichwere not seen in those from labeled whole cells.The surface peptide maps of Pllls from labeledwhole cells also have several peptides not seenin those from labeled OMs. These data indicatedthat the PIII molecule, like the PI molecule, hasa different exposure in OM of whole cells than inthe membrane of OM vesicles.

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FIG. 5. a-Chymotryptic surface peptide maps of Plll bands excised from an SDS-PAGE gel of lysates ofOMs of P- 0- N. gonorrhoeae strains JS1, JS2, JS3, and JS4 which were radioiodinated with lodogen. (A)Surface peptide maps of 30K PIlls (unreduced form of PIlls); (B) 31K PIII*a (upper band in doublet of reducedform of 31K PlIls resulting from Iodogen treatment); (C) 31K PIII*b (lower band in doublet of reduced form of31K Pllls resulting from lodogen treatment); (D) representative PIII chloro-T-125I-peptide map. This figure isincluded for comparison with the various PIII surface peptide maps. A\, Peptides which appeared to correspondto radioemitting peptides seen in surface peptide maps of Pll bands excised from an SDS-PAGE gel of lysates ofradiolabeled whole cells; A, Peptides which appeared to correspond to radioemitting peptides seen in surfacepeptide maps of PIll bands excised from an SDS-PAGE gel of lysates of radiolabeled OMs. TLE, Thin-layerelectrophoresis; TLC, ascending thin-layer chromatography.

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GONOCOCCAL SURFACE PEPTIDE MAPPING 639

As with the surface peptide maps of PIIIsfrom radiolabeled whole cells, those from la-beled OM vesicles were very similar in eachstrain. The doublet PIII*s, which occurred onlyin reduced lysates of Iodogen-radioiodinatedpreparations, had the same surface-exposedpeptides as the Pllls from unreduced lysates,indicating that the Iodogen reaction does notalter the surface exposure of PIII despite itseffect on migration of reduced PIIIs in SDS-PAGE.Based on the above observations, the follow-

ing conclusions could be drawn. (i) lodogen andLPO result in radioiodination of the same moi-eties of PI and PIII in whole cells. (ii) The PI ofeach N. gonorrhoeae strain has unique chymo-tryptic peptides exposed on the bacterial sur-face. However, the JS1 and JS2 PIs have severalshared surface peptides, whereas the JS3 andJS4 PIs, which share few, if any, surface pep-tides with JS1 and JS2, also share some surface-exposed peptides. (iii) The surface exposure ofPIs is much different in OM vesicles than inwhole cells. Virtually all of the peptides seen inchloro-T-125I-peptide maps of PIs could be seenin the surface peptide maps of PIs from radiola-beled OM vesicles, but the relative intensities ofthe peptides differed greatly in the two proce-dures, suggesting that PI may have a differentexposure in the membrane of OM vesicles thanit does in the OM of whole cells. (iv) The PIIIsfrom all four N. gonorrhoeae strains appeared tohave the same peptides exposed on the surfaceof whole cells regardless of the PI type withwhich it associated. (v) The PIlIs from all fourN. gonorrhoeae strains appeared to have thesame peptides exposed on the surface of OMvesicles. Two of these peptides appeared to bethe same PIII peptides exposed on the surface ofwhole cells. The remainder were different pep-tides, suggesting that the surface exposure ofPlll may be different in the membrane of OMvesicles than it is in the OM of whole cells.

DISCUSSIONIn this study, the nature of surface exposure

of PIs and PIIIs of four strains of N. gonor-rhoeae was explored by the technique of surfacepeptide mapping. Both intact whole cells andOM vesicles were radioiodinated with surface-reactive reagents. The radiolabeled PIs andPllls were isolated by SDS-PAGE and subjectedto a-chymotrypsic peptide mapping. The result-ant surface peptide maps of these proteins werethen compared with one another and withchloro-T-1 5I-peptide maps of exhaustively ra-dioiodinated (i.e., the chloramine-T procedure[3, 4]) PIs and PIlls.

In such a study, it is necessary to establishthat true surface labeling is accomplished.

Therefore, two radioiodination procedures werecompared. The LPO procedure relies on thebulk of the LPO enzyme (MW, ~=75,000 [17]) toexclude it from the cell, whereas the Iodogenprocedure employs an insoluble reagent (11)which is bound to the reaction vessel to preventinternal radiolabeling. If non-surface labelingwere occurring by either technique, the surfacepeptide maps derived from these proceduresshould have been markedly different. This wasnot the case, as can be seen in Fig. 2 and 4. Bothprocedures resulted in the same surface peptidesbeing labeled to the same relative intensity inboth the PI and PIII whole-cell surface peptidemaps. Therefore, it appeared that both proce-dures were predominantly labeling only exposedportions of the OM proteins.

Despite some similarities in radiolabeling byboth procedures, LPO was less efficient thanIodogen in labeling the PIII molecules. Thisphenomenon has been previously reported forseveral membrane proteins (11). The occurrenceof two distinct PIII bands in reduced SDS ly-sates which have been preradiolabeled with Io-dogen has been previously observed in our labo-ratory (unpublished data). The LPO procedurecaused smearing of the PIII band in reducedSDS lysates, suggesting that it too causes somemodification of PIII. Perhaps an explanation forthis can be found in a recent paper by McClard(12), who demonstrated that Iodogen cleavessulfhydryl groups from proteins. PIII surely hassulfhydryl groups (i.e., 2ME modifiable), andone of the two PIII bands seen in reducedlysates may have represented PIlIs which havelost sulfhydryl groups. LPO may also have re-moved sulfhydryls, albeit less efficiently thanIodogen, resulting in a smearing of PIII bands.The surface peptide maps of radiolabeled PIs

from whole cells demonstrated that proteinswhich have similar rimary structures (as seenin the chloro-T-1 I-peptide maps of JS1 PIversus JS2 PI or JS3 PI versus JS4 PI [Fig. 3Band reference 20]) can have different portions ofthe molecules exposed to the surface. It istempting to speculate that, since each PI exhib-its unique peptides on the surface, these por-tions will contain unique antigenic sites as well.As yet, the relationship between unique surfacepeptide maps and immunological specificity hasbeen definitively demonstrated for only two PIIsof N. gonorrhoeae JS1 (Swanson, Barrera, andJudd, manuscript in preparation). If this rela-tionship holds true for PIs as well, then severalinteresting predictions can be made.The similarity of the JS1 and JS2 PI surface

peptide maps suggested that antibody againstthese molecules might cross-react with bothstrains, whereas the similarity of the JS3 PI andJS4 PI surface peptide maps suggested that these

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INFECT. IMMUN.

proteins may cross-react with specific antiserato a greater extent than would the JS1 or JS2 PI.Supporting evidence for this speculation comesfrom radioimmunoprecipitation experimentswhich have shown that rabbit antiserum raisedagainst whole cells of N. gonorrhoeae JS1 isable to immunoprecipitate homologous PI andPlll but is unable to interact with the PI and PIIIof strain JS3 (21).The marked similarity of the PIII surface

peptide maps for all four strains would suggestthat these proteins would cross-react extensive-ly with strain-specific antisera. However, asdescribed above, antisera raised against strainJS1 was unable to immunoprecipitate either PIor PIII of strain JS3. It is therefore possible thatPIII is either non-immunogenic or is not antigen-ic in intact organisms, possibly reflecting a morehidden nature in PIII surface exposure.The similarity in surface exposure of PIII from

these strains would not seem unusual, since ithas been shown that these proteins have thesame primary structures (9), were it not for theobservations that PI and PIII form a heteropoly-mer in whole cells (15) and OM vesicles (13).Since the PI of each of the four strains studieddiffered in both primary structure and surfaceexposure, the same site on the PIII moleculesmust interact with a portion of the PI moleculewhich is common to all PIs. The exposure of thePIII molecule in the OM may, therefore, beindependent of the structural type of the PImolecule.The surface peptide maps of PIs and PIIIs

from radioiodinated whole cells correlated withthe sensitivity of these proteins to a-chymotryp-sin cleavage in situ (2). The JS1 and JS2 PIs,which have more chymotryptic surface peptidesthan do the JS3 or JS4 PIs, are susceptible tocleavage, whereas the JS3 PI, which has fewerchymotryptic surface peptides than does the JS1PI or JS2 PI, is resistant to in situ cleavage, as isthe JS4 PI (unpublished data). The PIII moleculeof all strains tested was resistant to chymotryp-tic cleavage in situ, which was possibly reflectedin the small number of peptides seen in PIIIsurface peptide maps. Since some surface pep-tides are generated by a-chymotrypsin cleavageof JS3 and JS4 PIs and PIIIs from all of thestrains which have been excised from SDS-PAGE gels, the chymotrypsin-sensitive sites onthe surface-exposed portions of these proteinsmust be protected from cleavage in situ perhapsby secondary, tertiary, or quaternary consider-ations.The difference between the surface peptide

maps of PI and PIlls from whole cells and thosefrom OMs is striking and suggests that, once amembrane bleb is freed from the cell, the expo-sure of the proteins in the membrane is altered.

The general patterns of similarities and differ-ences in surface exposure of PIs and PIlIs seenin the surface peptide maps of these proteinsfrom radiolabeled whole cells were also reflect-ed in the surface peptide maps derived fromradioiodinated OM vesicles.There appeared to be more of the PI molecule

available for surface iodination in the OM vesi-cles of all four strains than in whole cells. Therewere several peptides in the JS1 and JS2 PIsurface peptide maps which could not be direct-ly related to peptides in their chloro-T-1251-peptide maps (Fig. 2 and 3A, circles). It ispossible that these fragments are very weaklyiodinated, relative to other peptidic fragments,by chloramine-T and heavily iodinated by Iodo-gen. Also, chloramine-T has been shown tocause some cleavage of tryptophanyl peptidebonds (1), perhaps resulting in the loss or alter-ation of these fragments in the chloro-T-125I-peptide maps. The fact that essentially all of thepeptides seen in chloro-T-125I-peptide mapscould be visualized in the surface peptide mapsof PIs and PIlls (data not shown) suggests thatthere may be a range of surface exposure ofthese proteins in OM vesicles from a relativelysmall amount of exposure, as seen in wholecells, to virtually all of the protein. Alternative-ly, the Iodogen reagent may be disruptive to theisolated OM vesicle. Despite the cause, therewere more intensely emitting peptides in thesurface peptide maps of PIs from radiolabeledOM vesicles than there were in those fromradiolabeled whole cells, suggesting that boththe quantity and the quality of PI antigenicdeterminants may be different in OM vesiclesthan in whole cells. This observation may havedirect relevance to serotyping schemes whichuse OM vesicles as serotypic antigens (7, 8).

It appeared that PIII also had a differentsurface exposure in OM vesicles than in intactN. gonorrhoeae. As was the case in surfacepeptide maps of PIIIs from radioiodinated wholecells, PIIIs from radiolabeled OMs had similarsurface peptide maps regardless of the strainfrom which they were obtained. There were notmany more intensely emitting peptides in thesurface peptide maps of PIIIs from radioiodi-nated OM vesicles compared with those fromwhole cells; rather, there were several differentpeptides seen in these surface peptide maps. Theimmunological significance of this observationwill remain obscure until it is determined wheth-er PIlIs are antigenic in either whole cells orOMvesicles.The technique of surface peptide mapping

appears to be a useful tool in comparing thesurface exposure of OM proteins. Certainly,similarities and differences in surface exposurecan be demonstrated by this technique. By com-

64 JUDD

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GONOCOCCAL SURFACE PEPTIDE MAPPING 641

parison with chloro-T-125I-peptide maps, it canbe shown that primary structural differencesresult in different surface exposures. There isalso some evidence, as outlined above, to sug-gest that surface peptide maps may reflect im-munological reactivities. Techniques are cur-rently being developed to determine whichpeptidic fragments react with specific antibody.Further immunological data such as that gainedby radioimmunoprecipitation experiments willbe helpful in evaluating the surface exposure andantigenic determinants of the various OM pro-teins of N. gonorrhoeae.

ACKNOWLEDGMENTS

I thank Susan Smaus for her expert assistance in preparingthis manuscript and Chuck Taylor and Bob Evans for theirfine photographic work. I also am especially grateful to JohnSwanson for his excellent guidance and to the staff of theLaboratory of Microbial Structure and Function for theircritical evaluation and assistance in this work.

LITERATURE CITED

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2. Blake, M. S., E. C. Gotschilch, and J. Swanson. 1981.Effects of proteolytic enzymes on the outer membraneproteins of Neisseria gonorrhoeae. Infect. Immun.33:212-222.

3. Elder, J. H., R. A. Pickett II, J. Hampton, and R. A.Lerner. 1977. Radioiodination of proteins in single poly-acrylamide gel slices. J. Biol. Chem. 252:6510-6515.

4. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1%3.The preparation of 131I-labelled human growth hormone ofhigh specific radioactivity. Biochem. J. 89:114-123.

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

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

7. Johnston, K. H. 1978. Antigenic profile of an outer mem-brane complex of Neisseria gonorrhoeae responsible forserotypic specificity, p. 121-129. In G. F. Brooks, E. C.Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E.

Young (ed.), Immunobiology of Neisseria gonorrhoeae.American Society for Microbiology, Washington, D.C.

8. Johnson, K. H., K. K. Holmes, and E. C. Gotschlich.1976. The serological classification of Neisseria gonorr-hoeae. I. Isolation of the outer membrane complex re-sponsible for serotypic specificity. J. Exp. Med. 143:741-758.

9. Judd, R. C. 1982. 1251 peptide mapping of protein IIIisolated from four strains of Neisseria gonorrhoeae. In-fect. Immun. 37:622-631.

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

11. Markwell, M. A. K., and C. F. Fox. 1978. Surface-specif-ic iodination of membrane proteins of viruses and eucary-otic cells using 1,3,4,6-tetrachloro-3a,6a-diphenylglyco-luril. Biochemistry 17:4807-4817.

12. 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.

13. 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.

14. Morrison, M. 1980. Lactoperoxidase-catalyzed iodinationas a tool for investigation of proteins. Methods Enzymol.70:214-220.

15. Newhall, W. J., W. D. Sawyer, and R. A. Haak. 1980.Cross-linking analysis of the outer membrane proteins ofNeisseria gonorrhoeae. Infect. Immun. 28:785-791.

16. Philips, D. R., and M. Morrison. 1971. Exposed proteinon the intact human erythrocyte. Biochemistry 10:1766-1771.

17. Rombants, W. A., W. A. Schroeder, and M. Morrison.1967. Bovine lactoperoxidase. Partial characterization ofthe further purified protein. Biochemistry 6:2965-2977.

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

19. Swanson, J. 1978. Studies on gonococcus infection. XIV.Cell wall protein differences among color/opacity colonyvariants of Neisseria gonorrhoeae. Infect. Immun.21:292-302.

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