Vol. 38, No. 3INFECTION AND IMMUNITY, Dec. 1982, P. 898-9060019-9567/82/120898-09$02.00/0Copyright C 1982, American Society for Microbiology

Outer Membrane Characteristics of Campylobacter jejuniSUSAN M. LOGAN AND TREVOR J. TRUST*

Department ofBiochemistry and Microbiology, University of Victoria, Victoria, British Columbia, CanadaV8W 2 Y2

Received 7 May 1982/Accepted 30 August 1982

Outer membranes were isolated from type strains and wild-type isolates ofCampylobacter jejuni and Campylobacter coli by sodium lauryl sarcosinateextraction, and the polypeptide complement and lipopolysaccharide (LPS) con-tent were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The protein profiles exhibited by membranes from both species were quitesimilar, but could be distinguished from the type strain of the genus, C. fetussubsp. fetus CIP5396. The sodium dodecyl sulfate electrophoretograms of C.jejuni and C. coli were dominated by a major polypeptide band. In the referencestrain C. jejuni VC74, this polypeptide had an apparent molecular weight of45,000, was heat modifiable, and was shown to be transmembrane by virtue of itspeptidoglycan association and surface exposure. Two other proteins with approxi-mate molecular weights of 37,000 and 73,000 were also surface exposed on C.jejuni VC74 and represented potential surface antigens. The LPS of C. jejuni andC. coli was of low molecular weight, suggesting that serotypic differences due toLPS were based on different carbohydrate compositions of core LPS. In contrast,the LPS of C. fetus CIP5396 exhibited 0 antigen polysaccharide chains ofintermediate chain length. Fragments of outer membranes released during growthof C. jejuni VC74 displayed a polypeptide profile which differed from that ofsarcosinate-extracted outer membranes. Radiolabeling demonstrated that theproteins exposed on the surface of this released membrane differed from thoseexposed on the cell surface and would likely contribute to the antigenic complex-ity of C. jejuni.

The outer membrane (OM) of a gram-negativepathogenic bacterium serves as an interface be-tween the pathogen and its host and clearlyplays a most important role in the outcome ofhost-parasite relationships. Studies with a varie-ty of pathogens have shown that components ofthe OM can participate in adherence of thepathogen to host cells, invasion of host cells,resistance to the bactericidal activities of serum,resistance to phagocytosis and to phagocytickilling mechanisms, and sequestering of iron (2,11, 14). The outer leaflet of the lipid bilayer alsocontains lipopolysaccharide (LPS), whose lipidA component contributes the endotoxicity ofgram-negative cells (30). Moreover, the polysac-charide portion of the LPS and a number ofOMproteins can be exposed on the surface of thecell, and so serve as antigens (2, 4). Thesesurface-exposed constituents of the OM aretherefore important in determining the specific-ity of the immune response of the host and arealso key determinants in serotyping and bacteri-ophage typing schemes.Whereas the OM of a variety of gastrointesti-

nal pathogens of humans, including Escherichiacoli, Salmonella typhimurium, and Vibrio chol-

erae, have been well characterized (2, 4, 12), theOM of another very common gastrointestinalpathogen has received little attention. Campylo-bacterjejuni and its thermophilic relatives haverecently been recognized as significant patho-gens of man, producing an acute enteritis whichis often accompanied by a bacteremia (3, 31).There are also increasing reports of their in-volvement in systemic infections, includingmeningitis, endocarditis, and septicemia (27,37). Because of the inherent importance of theOM in the virulence of gram-negative bacteria,and because of current interest in the develop-ment of effective typing schemes for thermophil-ic campylobacters, we have characterized theprotein composition and topology and the LPSof the OM of representative strains of C. jejuniand Campylobacter coli, and here we report ourfindings.

MATERIALS AND METHODSBacterial strains and growth conditions. The Cam-

pylobacter strains examined in this study and theirsources are listed in Table 1. Stock cultures weremaintained at -70°C in 15% (vol/vol) glycerol-Trypti-case soy broth (BBL Microbiology Systems, Cockeys-

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TABLE 1. Campylobacter strains usedStraina Sourceb

C. coliCIP7080c Pig feces (CIP)C-35 Human feces (H. Lior)C-52 Human feces (H. Lior)C-343 Pig feces (H. Lior)8446 Human blood (M. B. Skirrow)

C. fetusCIP53%c,d Sheep fetus brain (CIP)

C. jejuniCIP702C Human feces (CIP)1123-47578 Human feces (S. McDermott)C-88 Human feces (H. Lior)VDP-3 Human blood (J. P. Butzler)J-2 Human feces (J. P. Butzler)Soton 5 Human feces (A. D. Pearson)VC74 Human feces (this laboratory)Penl Human feces (J. L. Penner)a The identity of the strains was determined by

DNA-DNA hybridization (R. J. Belland and T. J.Trust, J. Gen. Microbiol., in press).

b CIP, Collection de l'Institut Pasteur, Paris,France; H. Lior, Laboratory Center for Disease Con-trol, Ottawa, Ontario, Canada; M. B. Skirrow,Worcester Royal Infirmary, England; S. McDermott,Institute of Medical and Verterinary Science, Ade-laide, Australia; J. P. Butzler, St. Pierre Hospital,University of Brussels, Belgium; A. D. Pearson, Pub-lic Health Laboratory, Southampton, England; J. L.Penner, University of Toronto, Toronto, Ontario,Canada.

c Type strain.d Type species of Campylobacter.

ville, Md.). For membrane preparation, cultures weregrown in anaerobic jars for 36 h on Mueller-Hintonagar (Difco Laboratories, Detroit, Mich.). An atmo-sphere containing 5% oxygen and 10%O C02 was pro-duced with a gas generation kit for campylobacters(Oxoid Ltd., Basingstoke, England). After being har-vested, the cells were washed once in cold 20 mM Trisbuffer (pH 7.4). E. coli Cold Spring Harbor (CSH)strain 7 and S. typhimurium BS100-118 (this labora-tory) were also used.

Isolation of OMs. (i) SLS solublization. One gram(wet weight) of washed cells was resuspended in 10 mlof 20 mM Tris buffer (pH 7.4). DNase and RNase(Sigma Chemical Co., St. Louis, Mo.) were added,and the cell suspension was then passed three timesthrough a French press at 16,000 lb/in2. Intact cellswere removed by two centrifugations at 3,000 x g and4°C for 30 min. The total cell membrane fraction wasthen collected by centrifugation at 43,000 x g and 40Cfor 30 min. Cell membranes were resuspended in theTris buffer to give a protein concentration of 1 mg/mlof cell membrane preparation. OMs were prepared bydifferential solubilization of the inner membrane withthe detergent sodium lauryl sarcosinate (SLS) as de-scribed by Filip et al. (6). The total membrane protein/detergent ratio used was 1:6 (mg/mI). After beingshaken for 30 min at room temperature, the prepara-

tion was centrifuged at 43,500 x g for 30 min at 4°C,and the pellet was washed three times in the Trisbuffer.

(il) Isolation of rekased membrane fragments. OMmaterial released during growth was recovered bydifferential centrifugation. Cells (10 g) were suspendedin 100 ml of cold Dulbecco phosphate-buffered saline(pH 7.4) (Oxoid); whole cells were then removed bythree centrifugations at 5,000 x g for 10 min and thenone centrifugation at 12,000 x g for 15 min. Thesupernatant was then centrifuged at 5°C for 2.5 h at100,000 x g in a 50.2 Ti Beckman rotor. The resultantgel-like pellet was suspended in Tris buffer (pH 7.4),washed, and then stored at -10°C to be used.Chemical determinations. The protein content of the

membrane fractions was determined by the modifiedLowry procedure developed by Markwell et al. (17),with bovine serum albumin (Sigma) as the standard.LPS was assayed by determining 2-keto-3-deoxyoc-tonate by the method of Osborn et al. (24).SDS-PAGE. OM components were separated by

using the sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) system described byLaemmli (13). Proteins were stacked in 4.5% acrylam-ide and separated in 12.5% acrylamide. On occasion,separating gels of 7%, 12%, and 20% were used.Twenty to thirty micrograms of protein was appliedper lane. Gels were run at a constant current of 10 mAper gel until the tracking dye entered the separatinggel; the current was then increased to 20 mA. Proteinwas normally visualized with Coomassie blue, al-though some gels were silver stained by the method ofWray et al. (36). Carbohydrate was visualized with theperiodic acid-Schiff procedure of Fairbanks et al. (5).Some gels were stained with the cationic carbocyaninedye 1 - ethyl - 2 - [3 - [1 - ethylnaphtho(1 ,2d) - thiazolin - 2-ylidine] - 2 - methylpropanyl] - naphtho(1,2d)thiazoliumbromide (Stains-All; Eastman Kodak Co., Rochester,N.Y.) by the modified procedure of Green et al. (9)reported by Kelley and Parker (12). For autoradiogra-phy, gels were stained, destained, dried onto filterpaper, and overlaid with Kodak X-ray film.

32P radiolabeliag of LPS. For the preparation of 32p_radiolabeled membrane fractions, cells were grown onMueller-Hinton plates containing 150 LCi of 32p, (NewEngland Nuclear Corp., Boston, Mass.) (23). A smallsample was assayed for radioactivity to determine theextent of 32P radiolabeling. Strains of S. typhimuriumand Aeromonas salmonicida with defined alterationsin LPS structure were used as controls.

Radioiodination of surface proteins. Cells from a 36-hplate culture were washed and suspended in coldDulbecco phosphate-buffered saline. A 1:10 dilution ofthis gave an absorbance of 1.0 at 550 nm. A 100-,ulsample of the original suspension was then radioiodi-nated by using the New England Nuclear Corp. ra-dioiodination system (1251) which utilizes immobilizedlactoperoxidase and glucose oxidase. Enzyme beadswere removed by brief low-speed centrifugation, andthe '25I-labeled cells were then washed five times andsuspended in 20 mM Tris buffer (pH 7.4). OMs wereprepared from these cells.

Released OM fragments suspended to a proteinconcentration of 1 mg/ml were iodinated in a similarfashion.

Peptidoglycan-associated protein. The total cellmembrane was suspended in extraction buffer contain-

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MW

66 2 -

-!t .:4

45- -

_. ~1W

21 5

14-4

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 1. SDS-PAGE of isolated OM of strains of C.coli, C. fetus, Cjejuni, E.coli, and S.typhimurium.Lanes: 1, C. coli C343; 2, C. coli C602; 3, C. coli 8446;4, C. jejuni UDP-3; 5, C. jejuni 1123-47578; 6, C. jejuniSoton 5; 7, C. jejuni VC74; 8, C. coli CIP7080; 9, C.jejuni CIP702; 10, C. fetus CIP5396; 11, S. typhimur-ium BS100-118; 12, E. coli CSH7. MW, molecularweight (x 103); Lpp, lipoprotein.

ing 2% (wt/vol) SDS, 10 mM Tris (pH 7.4), and 10%o(vol/vol) glycerol and incubated at 60°C for 30 minaccording to the method of Rosenbusch (28). Proteinnot solubilized by this treatment is peptidoglycanassociated and was isolated by centrifugation at 45,000x g for 1 h; the pellet was then washed and suspendedin distilled water.

Electron microscopy. Unfixed specimens were nega-tively stained on Formvar-coated grids with 2% (wt/vol) phosphotungstic acid and examined in a PhillipsEM 300 electron microscope.

RESULTS

Protein compositions of OMs from Campylobac-ter spp. The SDS-PAGE protein profiles of OMfractions isolated from wild-type isolates andtype strains of C. coli and C. jejuni, as well asthe type species of the genus, C. fetus subsp.fetus CIP5396, are shown in Fig. 1. Also includ-ed for comparative purposes are OM profiles ofS. typhimurium BS100-118 and E. coli CSH7.The OM profiles of C. coli and C. jejuni couldreadily be differentiated from those of C. fetus,S. typhimurium, and E. coli. Whereas the pro-files of C. fetus, S. typhimurium, and E. coliwere dominated by two or more major proteinsin the molecular weight range of 33,000 (33K) to48K, the profiles of the thermophilic campylo-bacters were dominated by a single major pro-tein band. The molecular weight of this majorprotein varied between 43K and 45K dependingon the strain tested. In addition to this majorprotein, strains of C. coli and C. jejuni containedsix to eight other prominent OM proteins, al-though molecular weights and relative concen-trations varied from strain to strain. One bandwhich appeared to be common to all strains,including C. fetus, S. typhimurium, and E. coli,was a diffuse, lightly staining band at approxi-mately 20 kilodaltons. Another protein band wasclearly absent from the OM profiles of the

campylobacters. This was Brauns lipoprotein,which in its free form has a molecular weight of7,200 (1). In Fig. 1, Brauns lipoprotein can beseen in both the S. typhimurium and E. coliprofiles running just behind the dye front. Theidentity of this band was confirmed by stainingwith the lipid dye Oil Red 0 and with Stains-Alldye. C. fetus has previously been reported tolack this protein (18, 34).Because the protein profiles exhibited by the

various thermophilic campylobacters appearedto be relatively similar, a representative strainwas chosen as a reference for a more detailedmembrane characterization. The strain chosen,VC74, was a typical fecal isolate of C. jejuni.This strain was known to be virulent, havingbeen reisolated from a laboratory-acquired en-teritis. The SDS-PAGE protein profile of theOM of C. jejuni VC74 solubilized at 100°C isshown in Fig. 2A, lane 1. The seven majorprotein bands have been assigned letters for easeof description. The proteins exhibited the fol-lowing apparent molecular weights; band a,92K; band b (doublet), 75K; band c, 63K; bandd, 55K; band e, 45K; band f, 37K; band g, 20K.This profile was not altered with time of growth.Membranes prepared from cells grown for 18,24, 36, 48, and 72 h displayed the same SDS-PAGE profile (data not shown). When gels werestained with Stains-All dye, each band appearedorange-red. When gels were silver stained by theprocedure of Wray et al. (36), the major OMprotein (band e) stained very poorly, giving atransparent appearance. This staining character-istic was also seen with the 47-kilodalton proteinof C. fetus and the major pore-forming proteinsof both S. typhimurium and E. coli.Heat modifiability of OM proteins of C. jejuni

VC74. The OMs of a variety of gram-negativespecies contain heat-modifiable proteins; that is,when the OM preparation is heated above a

a's'Al*AMW

92 5 -66 2.-

45 -

31 -

a-- ab-

d-e-qof

MW

"*N" 6 6.2

_ W 45

A .....21 2 3

21 5- g-

1 2

FIG. 2. SDS-PAGE of isolated OM of C. jejuniVC74 solubilized for 3 min at different temperatures.Only those parts of the gel of interest are shown, andthe major proteins are labeled a-g. (A) Lane 1, 100°C;lane 2, 37°C. (B) Lane 1, 50°C; lane 2, 60°C; lane 3,700C. MW, molecular weight (x103).

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certain temperature in the presence of SDS, thepositions and numbers of protein bands on SDS-gel electrophoretograms are altered (4, 10, 19).When we examined the campylobacter OM forthis property, we found that the SDS-PAGEprofile of C. jejuni VC74 was influenced marked-ly by the temperature used to solubilize themembrane in SDS-2-mercaptoethanol. This wasespecially true of the electrophoretic mobilityand apparent molecular weight of the majorprotein (band e). When membranes were solubi-lized at 37°C (Fig. 2A, lane 2) much of thisprotein remained associated with oligomericcomplexes of molecular weight >92K. Theseprotein complexes gave a characteristic "wash-board" pattern. A significant amount of proteine ran with an apparent molecular weight of 33K,and a small amount electrophoresed with anapparent molecular weight of 45K. Solubiliza-tion at 50°C (Fig. 2B, lane 1) resulted in the lossof the high-molecular-weight complex, and themajority of protein e ran in the globular formwith the lower apparent weight (33K). At 60°C(Fig. 2B, lane 2), the majority of protein eappeared in the linear 45K form, and solubiliza-tion was complete at 70°C (Fig. 2B, lane 3).Membranes solubilized at temperatures of 50°Cand lower also displayed a protein with anapparent molecular weight of 40K (Fig. 2B, lane1). When membranes were solubilized at 60°C,this protein was not seen; rather, protein d (55K)appeared (Fig. 2B, lane 2). The 92K protein wasalso solubilized at 60°C (Fig. 2B, lane 2), where-as protein f was not solubilized until 100°C (Fig.2A, lane 1). No change in protein profile wasseen when membranes were solubilized at 100°Cin the absence of 2-mercaptoethanol, indicatingan absence of intrachain disulfide bridges in themajor OM proteins, or when membranes weresolubilized at 100°C for up to 60 min. Theaddition of ZnSO4 during membrane solubiliza-tion did, however, affect the heat modifiabilityof protein e. Hancock and Carey (10) havedemonstrated that when ZnSO4 is added duringsolubilization, the transition of OM protein Dlof Pseudomonas aeruginosa to its heat-modifiedform is promoted. We observed a similar effectwith protein e of C. jejuni VC74. In the presenceof 0.1 M ZnSO4, the transition to the linear formwas promoted at 45°C and complete at 60°C(data not shown).

Peptidoglycan association with the major OMprotein. The association of the OM proteins ofC. jejuni VC74 with the underlying peptidogly-can layer was investigated next. It is well knownthat the peptidoglycan-associated proteins of E.coli cannot be released from the peptidoglycanby heating the peptidoglycan-OM complex at60°C in 2% SDS; however, the proteins arereleased at 70°C (28). When the total cell mem-

MW

-92 5- --66-2

-031

- 21-5

1 2 3

FIG. 3. SDS-PAGE of peptidoglycan-associatedprotein and OM isolated from 125I-lactoperoxidase-labeled whole cells of C. jejuni VC74. Lanes: 1,peptidoglycan-associated protein stained with Coo-massie blue; 2, autoradiograph of isolated OM pre-pared from cells labeled with 1251-lactoperoxidase; 3,OM in lane 2 stained with Coomassie blue. MW,molecular weight (x103).

brane fraction of C. jejuni VC74 was treated at60'C by the method outlined by Rosenbusch(28), only protein e remained (Fig. 3, lane 1),indicating that this major OM protein was pepti-doglycan associated. The major OM protein ofC. coli C-35 was similarly shown to be peptido-glycan associated (data not shown).

Radioiodination of surface-exposed proteins.Since in the case of pathogenic bacteria, thoseproteins of the OM which are exposed on thecell surface may have important roles in thehost-parasite relationship, the surface exposureof proteins of the OM of C. jejuni VC74 wasdetermined. Cells were radiolabeled with 1251lactoperoxidase by an impermeant labeling pro-cedure, and OM isolated from these cells wasanalyzed by SDS-PAGE and autoradiography.Only three of the OM proteins were labeled (Fig.3, lane 2). The major protein e and the 76Kprotein b were both labeled much more stronglythan the 37K protein f. The effect of growthphase on the surface exposure of these threeproteins was also examined. All three proteinswere labeled on cells grown for 18, 24, 36, and 72h(data not shown).SDS-PAGE analysis of LPS composition. Anal-

yses of the LPS content of the OM of S. typhi-murium and E. coli have revealed that LPSmolecules of many sizes occur in the OM of asingle strain (8, 22, 25). The size heterogeneityof Campylobacter LPS was investigated bygrowing bacteria in the presence of 32P. Radiola-beled LPS in SDS-PAGE profiles of OM wasdetected by autoradiography, whereas the pres-ence of carbohydrate was confirmed by staininggels with periodic acid-Schiff reagent and Stains-All dye. The results in Fig. 4 reveal the sizeheterogeneity in Campylobacter LPS. The sim-plest LPS was that of the reference strain C.

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A MW B

-W*- 92 5 -

662 -

_i- 45 -

- 31 .

21 5*o

14 4 _

_0 vPL * *

X 2 3

FIG. 4. SDS-PAGE ofOM isolated from cells of strains of C. coli, C. fetus, and C. jejuni grown with 32P. (A)OM isolated from C. jejuni VC74. Lanes: 1, OM solubilized at 30°C for 3 min and stained with Coomassie blue; 2,autoradiograph ofOM in lane 1; 3, autoradiograph ofOM solubilized at 100°C for 3 min; 4, OM in lane 3 stainedwith Coomassie blue. (B) OM isolated from C. fetus CIP5396. Lanes: 1, OM solubilized at 30°C for 3 min andstained with Coomassie blue; 2, autoradiograph ofOM in lane 2; 3, autoradiograph ofOM solubilized at 100°C for3 min; 4, OM in lane 3 stained with Coomassie blue. (C) Autoradiographs of OM isolated from C. jejuni C-88(lane 1), C. coli C-52 (lane 2), C. coli C-343 (lane 3), and C. jejuni Penl (lane 4). MW, molecular weight (x103).PL is phospholipid.

jejuni VC74 (Fig. 4A, lane 3) and C. coli C-343,in which the labeled LPS migrated as a singleclass in the approximate molecular weight range(with respect to protein) of 14K to 21.5K. TheLPS of C. jejuni C-88 and C. coli C-52 (Fig. 4C,lanes 1 and 2, respectively) displayed two class-es of LPS as demonstrated by the intensity ofthe radiolabel. This was most evident in the case

of C. jejuni C-88, in which both classes were

smaller. The LPS of C. jejuni Penl was some-what more complex, showing an even smallerclass of LPS than did C. jejuni C-88.The LPS content of C. fetus CIP5396 was

more heterogeneous than that of the strains of C.coli and C. jejuni examined. Membranes of C.fetus solubilized at 100°C (Fig. 4B, lane 2) dis-played an LIPS class of intermediate size whichmigrated in the approximate molecular weightrange (with respect to protein) of 24K to 31K.LPS also appeared to be associated with a 65Kprotein in the OM of C. fetus CIP5396. A smallamount of radiolabel was similarly associatedwith the 63K protein c (putative flagellin) of C.jejuni C-74, although this was better visualizedwhen the film was exposed for a longer time thanthat shown in Fig. 4A, lane 2. Radiolabel was

also associated with a corresponding protein inC. jejuni C-88 (Fig. 4C, lane 1) and C. coli C-343.In the latter case this association was again moreapparent when the film was exposed for a longertime than that shown in Fig. 4C, lane 3. Fig. 4also shows the effect of the temperature ofSDS-2-mercaptoethanol solubilization on the electro-phoretic mobility of Campylobacter LPS. At a

30°C solubilization temperature, much of theLPS remained associated with the high-molecu-

lar-weight protein "washboard" complex. Thisis best illustrated by C. fetus CIP5396 in Fig. 4B,lanes 1 and 2. Indeed, in the case of C. jejuniVC74 (Fig. 4A, lanes 1 and 2, respectively),much of the LPS-protein complex was so largeat the 30°C solubilization temperature that itremained behind in the stacking gel, and only a

small amount of protein and radiolabeled LPSpenetrated the separating gel.SDS-PAGE analysis of released OM fragments.

A wide variety of gram-negative bacteria pro-

duce OM evaginations or blebs during growth.Many of these blebs are subsequently releasedinto the surrounding milieu as OM vesicles or

fragments (16, 20, 21). This released materialmay be an important form offree endotoxin (29).Electron microscopic examination of negativelystained preparations of 36-h cultures of C. jejuniVC74 revealed the presence of much bleb andfragment material. Released OM fragments weresubsequently isolated by differential centrifuga-tion and analyzed by SDS-PAGE. The proteinprofile of the released OM differed from that ofOM isolated by SLS extraction (Fig. 5). Proteinsa, c, and e were present in the vesicle fraction,with considerable enrichment of protein a. OMproteins b, d, e, f, and g were also present inmarkedly reduced amounts in the OM fragmentsand were only visualized by overloading thegels. The vesicle fraction also contained as a

major constituent a 12K protein. This bandsometimes stained poorly with Coomassie blueand, as indicated by the arrow in Fig. 5A, lane 1,could be seen associated with a fast-migrating,wavy band. This wavy character was due to thepresence of LPS in that region of the gel. The

C-; MW

g

1 2 3 4

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I 2 :". .1%

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92 * § - ^... s.....-3162

455-

31 - ..

215 -

14 4-

*0 *-

Lpp-

1 2

FIG. 5. SDS-PAGE of rments of C. jejuni VC74. (Abrane fragments stained withautoradiograph of releasedbelled with 125I-lactoperoxidmembrane; lane 2, OM; lanfragments. (C) Lane 1, OMreleased membrane fragmentwere stained with Coomassareas of the gel of interest ar

weight; Lpp, lipoprotein; arr

membrane fragments but noi

12K protein was better v

were overloaded (Fig. 51present in OM preparatioit was present in total ctions (Fig. 5B, lane 1). Siiproteins are largely lostthe total cell membrane frwould appear to be ass

membrane. This proteinthe free form of Brauns(Fig. 5C).The topological distrib

the released OM fragmened by radiolabeling with ttechnique previously appprotein a, which had nonative membrane, was IOM fraction (Fig. SA, lateins, OM protein e andwere radiolabeled. Thisprotein, which stainedblue, was strongly labepresent in the OM fragirvisualized with this techi

DISCUSThis examination of tyI

isolates of C. jejuni andthey possess quite simil;and that the two species con the basis of their SD'

B MW In contrast, both species could be distinguished4 _ X 1from the type species of the genus, C. fetus

l a~. subsp. fetus CIP5396. TheOM protein profile oft 7t *w-31 C. jejuni and C. coli was characterized by the; ;'~~ -21^5 presence of a single major protein band. In-st_14.4 contrast, the OM protein profile of the type

strain of C. fetus displayed two major bands.This was in general agreement with the findings

1 2 3 of McCoy et al. for C. fetus (18); however,whereas these workers reported molecular

C weights of 39K and 41K for the major C. fetusM_̂MW OM proteins, in our experimehts these proteins

-14.4 had apparent molecular weights of45K and 47K.These sizing discrepancies probably arise from

2* differences in methodology.1 2 In the reference strain C. jejuni VC74, the

released membrane frag- major protein spanned the membrane, beingi) Lane 1, released mem- exposed on the cell surface and also beingCoomassie blue; lane 2, associated with the peptidoglycan on the innermembrane fragments la- surface of the OM. The major OM protein of C.ase. (B) Lane 1, total cell jejuni VC74 was also heat modifiable and ap-ie 3, released membrane peared to be capable of associating in oligomers.of E. coli CSH7; lane 2, These findings suggest that this protein (band e)ts. The gels in (B) and (C) is the porin or matrix protein of C. jejuni,sie blue, and only those maintaining hydrophilic, size-dependent diffu-e shown. MW, molecular

sion channels through the OM. As such, thisow, band seen in released protein would obviously play an important rolein the nutrition of this pathogen, and the largeamount present in the OM (>70%o based on

isualized when the gels Coomassie blue staining) suggests an importantB, lane 3) and was not structural role as well.Ins (Fig. 5B, lane 2), but In C. jejuni VC74, this putative porin proteinell membrane prepara- had an apparent molecular mass of 45 kilodal-nce soluble periplasmic tons. This subunit size places it among theduring the isolation of largest of reported porins. Indeed, the largestraction, the 12K protein confirmed porin protein is the 42-kilodalton pro-ociated with the inner tein f of P. aeruginosa PA01 (10). This proteindid not correspond to forms porin channels of a larger diameter thanlipoprotein in E. coli those of E. coli or S. typhimurium, allowing for

an exclusion limit of 3,000 to 9,000 daltons,ution of the proteins in compared with the 600 daltons of the Enterobac-its was then investigat- teriaceae porins (4). It is conceivable that the:he impermeant labeling 43K to 48K transmembrane proteins of C. jejuni,lied to whole cells. OM C. coli, and C. fetus may also produce large-)t been radiolabeled in diameter pore channels. This would seem quitelabeled in the released advantageous for nutritionally fastidious orga-ne 2). Two other pro- nisms such as Campylobacter spp., since largerthe 12K protein, also pores should allow more ready access of largerlow-molecular-weight molecules such as peptides and oligonucleotides

poorly by Coomassie to the periplasmic space.led, and the quantity Surface labeling studies also revealed consid-nents was more clearly erable surface exposure for this major OM pro-nique. tein, implying an important contribution to the

iSION antigenic makeup of this pathogen. Certainly thestrain-to-strain size differences in this protein

pe strains and wild-type are likely to provide strain-to-strain antigenicC. coli has shown that differences. In the case of C. jejuni VC74, twoar OM protein profiles other OM proteins were surface exposed and:annot be differentiated must also contribute to the antigenic complexityS electrophoretograms. of the cell surfaces. Indeed, based on the rela-

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tive amounts of the proteins in Coomassie blueprofiles, more of the 74-K protein b moleculewould appear to be surface exposed than proteine. Preliminary findings with other strains (Loganand Trust, unpublished data) show that otherOM proteins can be surface exposed and willalso contribute to strain antigenic differences.Clearly the contributions made by these varioussurface proteins in serotyping schemes usingheat-denaturable antigens (15) now need to bedefined. One surface protein that this study didnot identify was the microcapsular protein of C.fetus. In the case of virulent strains of C. fetus, a98K glycoprotein which confers antiphagocyticproperties has been reported (35). Although wefound LPS associated with a 63K to 65K proteinin both C. fetus CIP5396 and C. jejuni VC74,there was no indication of the presence of amajor surface-exposed glycoprotein with a mo-lecular weight of 98K on C. jejuni VC74.Another major surface antigen of C. jejuni and

C. coli is LPS, and a typing system based onheat-stable antigens has been reported (26). OnSDS-PAGE, LPS migrates with lipid A presentand separates on the basis of size (8, 22, 25).Migration distance is inversely related to poly-saccharide content, so the core LPS with itsshort carbohydrate side chain migrates mostrapidly, whereas LPS with long 0 antigens mi-grates slowest. The technique is quite discrimi-nating and has been shown to separate LPSmolecules differing by as few as two or threesaccharides (8, 22, 25). Unlike the LPS of wild-type E. coli, S. typhimurium, and A. salmoni-cida previously examined by this technique (8,22, 23, 25), the LPS of wild-type C. jejuni and C.coli appears to be of low molecular weight andlacking in 0 antigens. In the case of C. jejuniVC74 and C. coli C-343, the LPS would appearto be simply composed of core-lipid A. In thecase of C. jejuni C-88, C. coli C-52, and C. jejuniPenl, this low-molecular-weight LPS has in-creased heterogeneity, with the core-lipid A ofC. jejuni being noticeably smaller. In these cas-es, the low-molecular-weight LPS might includeone repeating 0 chain unit attached to a com-plete core (22). One of the strains examinedhere, C. jejuni Penl, is in fact a reference strainin the serotyping system based on heat-stableantigens, and this LPS serogroup is one of thefour most common serogroups associated withhuman infections. Each of the other strainsexamined for LPS content belongs to differentheat-stable antigen serotypes (26). It would ap-pear that the serotyping of C. jejuni and C. coliLPS has a different basis than that of Salmonellaspp. That is, the serological differences betweenstrains of C. jejuni and C. coli are not due to longpolysaccharide chain 0 antigens but are due todifferences in the carbohydrates associated with

the low-molecular-weight core LPS. In contrastto C. jejuni and C. coli, the LPS of C. fetusCIP5396 did appear to have 0 antigen sidechains of intermediate chain length.

Like other gram-negative bacteria, campylo-bacters release OM during growth. Studies on E.coli have suggested that this OM material that isreleased by growing cells probably arises fromzones of OM growth called insertion regions,since the released OM is enriched in newlysynthesized proteins and LPS (7, 32, 33). Wen-sink and Witholt (33) have propoged that whenthe OM expands more rapidly than the underly-ing peptidoglycan layer, those areas of mem-brane not attached to the peptidoglycan will tendto bulge and continue to grow until released. Ifouter and inner membranes adhere at insertionregions, some cytoplasmic membrane materialmay be dragged into nascent vesicles. The mod-el of Wensink and Witholt also proposes that thereleased membrane is sealed off in the form of avesicle and that the sealed vesicles need not beempty but could carry periplasmic proteins in-side. In the case of C. jejuni VC74, the releasedOM fraction displayed a protein profile differentfrom that of OM prepared by SLS extraction.This altered profile appeared not to result fromdifferences in isolation methodology since SLS-extracted OM displayed the same profile afterisopycnic centrifugation in sucrose (Logan andTrust, unpublished data). Rather, the alteredprotein profile of the released OM fraction isconsistent with regions for preferential mem-brane release. Indeed, the reduced relativeamount ot protein e in the released membranefraction is consistent with it performing an im-portant role in the attachment of the OM to theunderlying peptidoglycan, whereas the presenceof a sizeable quantity of a protein associatedwith the inner membrane is consistent with theincorporation of some inner membrane into thenascent vesicle. Indeed, this 12K protein mayrepresent a specific adhesion zone protein.However, the surface radiolabel data indicatethat the released membrane does not have thesame orientation as native membrane. Protein ewas clearly exposed on the surface of releasedmembrane, but was not exposed on the surfaceof whole cells. Moreover the low-molecular-weight protein which was not present in OMpreparations was strongly surface exposed onreleased membrane. This suggests that themechanism of membrane release in C. jejuniVC74 is different from that in E. coli, and thereleased membrane may be inside out. Regard-less of the mechanism for release, the releasedOM fragments will clearly present an antigenicprofile which is different from that of the intactcell. The released OM fragments may also con-tribute to the pathogenesis of Campylobacter

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infections, by virtue of their inherent endotoxici-ty (Logan and Trust, unpublished data) or bydelivery of toxins to intestinal cells. It has beensuggested that OM vesicles play an importantrole in the delivery of heat-labile enterotoxinproduced by enteropathogenic strains of E. coli(20).

Studies are currently under way to confirmthe immunogenicity of the proteins exposed onthe surface of whole cells and released OMfragments and to identify and characterize theimmunological determinants. We hope thatthese studies will provide a clearer understand-ing of the contribution of membrane proteins tothe immunogenicity of thermophilic campylo-bacters.

ACKNOWLEDGMENTSThis investigation received financial support from the World

Health Organization. S.M.L. was the recipient of a Universityof Victoria Graduate Student Fellowship.

Henrik Chart provided valuable technical advice.

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