Vol. 55, No. 3INFECTION AND IMMUNITY, Mar. 1987, p. 828-8310019-9567/87/030828-04$02.00/0Copyright © 1987, American Society for Microbiology

Demonstration of a Flagellar Antigen Shared by a Diverse Group ofSpiral-Shaped Bacteria That Colonize Intestinal Mucus

ADRIAN LEE,t* SUSAN M. LOGAN, AND TREVOR J. TRUSTDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia,

Canada V8W2Y2

Received 3 June 1986/Accepted 17 November 1986

Western blot analysis showed that there is little immunological cross-reactivity of the human pathogensCampylobacter jejuni and Campylobacter coli with Campylobacter pyloridis, except for a very strong cross-

reaction between the flagellins. This same antigenic cross-reaction was found with two isolates of gram-negativespiral microaerophilic bacteria that are known to colonize the intestinal mucosa of rodents, but not with theflagellins of a number of other motile bacteria. It is proposed that this shared flagellin antigen may be importantstructurally and functionally.

The surfaces of the intestinal tract are normally colonizedby large numbers of bacteria that have evolved sophisticatedmechanisms of association that allow them to withstandperistalsis and the flow of intestinal chyme. Of particularinterest to us have been the organisms that are adapted tosurvive in the intestinal mucus of normal animals. Thesebacteria all have a spiral morphology, are microaerophilic,and can penetrate deeply into intestinal crypts (7). Recently,our work has included the study of three spiral-shapedmicroaerophilic human pathogens, i.e., Campylobacterjejuni and Campylobacter coli, which cause gastroenteritis(14), and Campylobacter pyloridis, which has been impli-cated as a cause of gastritis (2). We have proposed that'theseorganisms colonize human intestinal mucus in a mannersimilar to that of the spiral bacteria of the normal microbiota(4, 9).

In a study investigating the relationships between thesebacteria, a surprising and interesting immunological cross-reaction was found. Whole-cell lysates of C. jejuni, C. coli,and C. pyloridis together with lysates of two as yet unclas-sified spiral isolates, ST1 and Ti, from the rodent intestinalmucosa were prepared by boiling the lysates in the solubili-zation buffer described by Laemmli (6) and were separatedby the sodium dodecyl sulfate-polyacrylamide gel electro-phoresis system described by Laemmli (6). Electrophoreti-cally separated cell constituents on the 12.5% acrylamideslab gel were then electroblotted to nitrocellulose paper (15)and reacted with antiserum SML1 prepared in rabbitsagainst formalinized cells of C. jejuni VC74 (10). Thisantiserum had previously been shown to contain antibodiesto cross-reactive epitopes of a number of surface-exposed C.jejuni, C. coli, and Campylobacter fetus proteins, includingthe flagella and major outer membrane protein (11, 12).Bound antibody was revealed by using radioiodinated 12511labeled staphylococcal protein A and autoradiography. Asexpected, antibodies to many of the polypeptides of the C.jejuni and C. coli strains tested were present (Fig. 1A, lanes3 and 4), including the 63,000-molecular-weight (MW)flagellin (Fig. 1A, lane 2). In contrast, there was surprisinglylittle cross-reaction with the components of the C. pyloridis

* Corresponding author.t Present address: School of Microbiology, University of New

South Wales, Sydney, Australia 2033.

strains tested (Fig. 1A, lanes 5 and 6) or with the two rodentspiral bacteria (Fig. 1A, lanes 7 and 8). The exception was astrong reaction with a polypeptide that appeared to corre-spond to the C. jejuni and C. coli flagellins and that had anapparent MW of 58,000 to 59,000 depending on the straintested.

Morphologically, the four spiral organisms showing thisantigenically cross-reactive polypeptide were very different;they were selected for this investigation because they wereall microaerophilic, were spiral shaped, and colonized gas-trointestinal mucous surfaces. The characteristic morpholo-gies of these bacteria and examples of their mucous habitatsare shown in Fig. 2. The human pathogen C. jejuni has asingle unsheathed polar flagellum and, as we have shown,can colonize intestinal mucus in mice (9). In biopsy speci-mens from human stomach, C. pyloridis is seen to colonizemucus, but it is a very different organism, with sevensheathed flagella. The rodent isolates are also very different.Organism ST1 naturally colonizes ileal crypts in very largenumbers; has a very characteristic morphology, with 13 to 15sheathed flagella; and is surrounded with several concentricperiplasmic fibrils. We have suggested that ST1 belongs to anew genus of bacteria (13). Organism Ti, isolated frommouse cecum and as yet unidentified, looks more like C.jejuni but is a much longer spiral organism similar to thosecolonizing cecal crypts and has a single sheathed flagellum.To provide further evidence that the antigenically cross-

reactive polypeptide detected by antiserum SML1 wasflagellin, polyclonal antiserum SML2 prepared against mo-nomeric C. jejuni VC74 flagellin was used. The immunogenwas prepared by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis of purified flagella and electroblotting of thelinearized subunits to nitrocellulose paper. Antiserum SML2has been shown to be monospecific for internal conservedCampylobacter flagellin epitopes (12) and therefore reactedstrongly with C. jejuni VC185 and C. coli VC167 flagellins(Fig. 1B, lanes 1 through 4). Similar strong immunoblotreactions were obtained with the 57,000- to 59,000-MWpolypeptides of C. pyloridis 5155, C. pyloridis 5442, andspiral isolates ST1 and Ti, which had reacted with antiserumSML2 (Fig. 1B, lanes 5 through 8), providing strong evi-dence that in all cases the cross-reactive polypeptide wasflagellin. In contrast, no reaction was shown with anyproteins in whole-cell lysates prepared from motile cells of

828

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FIG. 1. Antigenic cross-reactivity demonstrated by Western blot

analysis. (A) Antiserum SML1 prepared against formalinized cells

of C. jejuni VC74 and reacted at a 1:100 dilution. Sodium dodecyl

sulfate-polyacrylamide gel electrophoresis of purified VC74 flagellin

stained by Coomassie blue (lane 1); autoradiograms of Western blots

of purified VC74 flagellin (lane 2) and of whole-cell lysatesof C.

jejuni VC185 (lane 3), C. coli VC167 (lane 4), C. pyloridis 5155 (lane

5), C. pyloridis 5442 (lane 6), rat intestinal isolate ST1 (lane 7), and

mouse intestinal isolate Ti (lane 8). (B) Antiserum SML2 prepared

against purified monomeric C. jejuni VC74 and reacted at a 1:100

dilution. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

of purified C. jejuni VC185 flagellin stained by Coomassie blue (lane

1); autoradiograms of Western blots of purified VC185 flagellin (lane

2) and of whole-cell lysates of C. jejuni VC185 (lane 3), C. coli

VC167 (lane 4), C. pyloridis 5155 (lane 5), C. pyloridis 5442 (lane 6),

rat intestinal isolate ST1 (lane 7), mouse intestinal isolate Ti (lane

8), V. cholerae MW13 (lane 9), A. hydrophila Al (lane 10), E. coli B2

(lane 11), S. typhimurium B4 (lane 12), Proteus vulgaris B5 (lane 13),

and Pseudomonasfluorescens B6 (lane 14). (C) Convalescent-stage

antiserum CP10 taken from a patient with naturally acquired C.

jejuni VC185 gastroenteritis and reacted at a 1:100 dilution. Auto-

radiograms of Western blots of purified C. jejuni VC185 flagellin

(lane 1) and of whole-cell lysates of C. pyloridis 5443 (lane 2), C.

pyloridis 5442 (lane 3), C. pyloridis 5155 (lane 4), C. jejuni VC185

(lane 5), rat intestinal isolate ST1 (lane 6), and mouse intestinal

isolate Ti (lane 7). (D) Antiserum ALl prepared against formalin-

ized cells of C. pyloridis 5155 and reacted at a 1:10 dilution.

Autoradiograms of Western blots of purified C. jejuni VC74 flagellin

(lane 1) and of whole-cell lysates of C. jejuni VC 74 (lane 2), C.

pyloridis 5155 (lane 3), C. pyloridis 5294 (lane 4), rat intestinal isolate

ST1 (lane 5), and mouse intestinal isolate Ti (lane 6). MWs are given

on the right in thousands.

Vibrio cholerae MW13, Aeromonas hydrophila Al, Esche-richia coli B2, Salmonella typhimurium B4, Proteus vulgarisB5, or Pseudomonas fluorescens B6 (Fig. 1B, lanes 9through 14).Immunoblot analysis with human convalescent-stage an-

tiserum obtained from a patient who had suffered a naturallyacquired gastroenteritis provided further evidence that thecross-reactive epitopes detected with antisera SML1 andSML2 were on flagellin. The strain responsible for thegastroenteritis was C. jejuni VC185, and the results of theanalysis (Fig. 1C) show the reaction of the convalescent-stage antiserum with purified C. jejuni VC74 flagellin (lane 1)and with polypeptides of corresponding MW in whole-celllysates of C. pyloridis 5443, 5442, and 5155 (lanes 2 through4) and of spiral isolates ST and Ti (lanes 6 and 7). Inwhole-cell lysates of homologous strain C. jejuni VC185,flagellin was the only antigen detected by this technique atthe 1:100 dilution of antiserum used (lane 5). No reactionwas observed in control assays of whole-cell lysates ofmotile V. cholerae.To further examine the immunologic cross-reactivity of

this group of spiral organisms, rabbits were immunized withformalinized whole cells of C. pyloridis 5155 to provideantiserum ALl. This antiserum also gave a strong reactionwith purified C. jejuni VC74 flagellin (Fig. 1D, lane 1).Indeed, when antiserum ALl was reacted with whole-celllysates of VC74, the 63,000-MW flagellin monomer was theonly antigenically cross-reactive polypeptide (Fig. 1D, lane2). With lysates of all other organisms tested, including thehomologous C. pyloridis 5515, the predominant antigen wasthe 57,000- to 59,000-MW polypeptide that also displayedantigenic cross-reactivity with antiserum SML1 and themonospecific antiflagellin antiserum SML2. Interestingly,antiserum ALl did show strain serospecificity, as can beseen by the broad antibody-antigen reaction at the front ofthe gel (lane 3) resulting from anti-lipopolysaccharide anti-body.

The, biological significance of the antigenic cross-reactivityof these flagellins is unclear at this time. However, organ-isms that colonize intestinal crypts or gastric pits are allactively motile spirals. This spiral shape, together with themotility conferred by their flagella, presumably gives them aselective advantage in the viscous intestinal mucus. Indeed,it has been proposed that for certain pathogens, includingCampylobacter species, this adaptation to the milieu ofintestinal mucus is an important determinant of pathogenic-ity (13). Colonization of mucus, particularly by organismsthat can move freely in it, allows close association with theintestinal surface, and, thus, the possession of specificadhesins is not required (8, 9). The possession of commonblot-reactive internal antigenic determinants by all of theseorganisms that colonize mucus implies conservation of afunctionally important portion of the flagellin molecule.Ibrahim et al. (5) have observed similar antigenic cross-reactivity among denatured monomeric flagellins in mem-bers of the Enterobacteriaceae. However, the cross-reactiveflagellin epitopes in the Enterobacteriaceae do not appear tobe antigenically related to the cross-reactive Campylobacterflagellin epitopes studied here, as antiserum SML2 showedno reaction with flagellins in the Enterobacteriaceae. Asnucleotide sequencing has shown considerable structuralconservation in both the N- and C-terminal sequences ofSalmonella (16), Bacillus (1), and Caulobacter (3) flagellinsand as N-terminal amino acid sequence analysis of C. jejuniand C. coli flagellins shows that they also share considerablesequence homology with these flagellins (L. A. Harris, S. M.

VOL. 55, 1987

830 NOTES

0.

IL -tj~~

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ipml 4im~2pm 1mFIG. 2. In vivo localization and in vitro morphology of four species of bacteria with antigenically cross-reactive flagellins. (a) Homologous

species C. jejuni. Scanning electron micrograph of surface mucus on the cecal mucosa of an antibiotic-treated mouse fed pure cultures ofhuman strain VC185, showing large numbers of the spiral organism in the mucus blanket. (Inset) Single cell from a pure culture. (b) C.pyloridis. Transmission electron micrograph of the gastric epithelium of a patient with gastritis, showing the spiral bacterium in the mucusand in proximity to goblet cells. (Inset) Single cell from a pure culture. (c) Rat intestinal isolate ST1. Transmission electron micrograph of ilealepithelium of a normal rat, showing the bottom of a mucus-filled crypt packed with ST1. (Inset) Single cell from a pure culture. (d) Mouseintestinal isolate Ti. Transmission electron micrograph of the cecal epithelium of a normal mouse, showing organisms morphologically similarto Ti at the opening of an intestinal crypt. (Inset) Single cell from a pure culture.

Logan, and T. J. Trust, unpublished data), the structuraldifferences giving rise to the antigenic differences betweenflagellin species probably reside in the amino acid sequencesmaking up the central portions of the flagellin molecules.

We thank Tanya Robertson for the isolation of Ti and SharonCubbage for skilled technical assistance.

This work was supported by the National and Medical ResearchCouncil of Australia, the Medical Research Council of Canada, andthe Natural Sciences and Engineering Research Council of Canada.

LITERATURE CITED

1. DeLange, R. J., J. Y. Chang, J. H. Shaper, and A. N. Glazer.1976. Amino acid sequence of flagellin of Bacillus subtilis 168.III. Tryptic pedtides, N-bromosuccinimide peptides, and the

complete amino acid sequence. J. Biol. Chem. 251:705-711.2. Eldridge, J., A. M. Lessels, and D. M. Jones. 1984. Antibody to

spiral organisms on gastric mucosa. Lancet i:1237.3. Gill, P. R., and N. Agabian. 1983. The nucleotide sequence of

the Mr = 28,000 flagellin gene of Caulobacter crescentus. J.Biol. Chem. 258:7395-7401.

4. Hazell, S. L., A. Lee, L. Brady, and W. Hennessy. 1986.Campylobacter pyloridis and gastritis: association with intercel-lular spaces and adaption to a mucus environment as importantfactors in colonization of the gastric epithelium. J. Infect. Dis.153:658-663.

5. Ibrahim, G. F., G. H. Fleet, M. J. Lyons, and R. A. Walker.1985. Immunological relationship between Salmonella flagellinsand between these and flagellins from other species of Entero-bacteriaceae. Med. Microbiol. Immunol. 174:101-103.

6. Laemmli, U. K. 1970. Cleavage of structural proteins during the

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f::,. k.,,;s..!:f , :. 10

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VOL. 55, 1987 NOTES 831

assembly of the head of bacteriophage T4. Nature (London)227:680-685.

7 Lee, A. 1984. Neglected niches. The microbial ecology of thegastrointestinal tract, p. 115-162. In K. C. Marshall (ed.),Advances in microbial ecology, vol. 8. Plenum PublishingCorp., New York.

8. Lee, A. 1985. The spiral-shaped inhabitants of intestinal mucosain normal and diarrhoeal humans and animals: pointers to noveldeterminants of pathogenicity, p. 181-185. In S. Tzipori (ed.),Infectious diarrhoea in the young. Excerpta Medica, Amster-dam.

9. Lee, A., J. L. O'Rourke, P. J. Barrington, and T. J. Trust. 1986.Mucus colonization as a determinant of pathogenicity in intes-tinal infection by Campylobacterjejuni: a mouse cecal model.Infect. Immun. 51:536-546.

10. Logan, S. M., and T. J. Trust. 1982. Outer membrane charac-teristics of Campylobacterjejuni. Infect. Immun. 38:898-906.

11. Logan, S. M., and T. J. Trust. 1983. Molecular identification of

surface protein antigens of Campylobacter jejuni. Infect. Im-mun. 42:675-682.

12. Logan, S. M., and T. J. Trust. 1986. Location of epitopes onCampylobacterjejuni flagella. J. Bacteriol. 168:739-745.

13. Phillips, M. W., and A. Lee. 1983. Isolation and characterizationof a spiral bacterium from the crypts of the rodent gastrointes-tinal tract. Appl. Environ. Microbiol. 45:675-683.

14. Skirrow, M. B. 1984. Campylobacter infections of man, p.105-141. In C. S. F. Easman and J. Jeljaszewicz (ed.), Medicalmicrobiology, vol. 4. Academic Press Inc. (London), Ltd.,London.

15. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:43504354.

16. Wei, L.-N., and T. M. Joys. 1985. Covalent structure of threephase-1 flagellar filaments proteins of Salmonella. J. Mol. Biol.186:791-803.