INFECTION AND IMMUNITY, Aug. 1990, p. 2686-2689 0019-9567/90/082686-04$02.00/0 Copyright 33 1990, American Society for Microbiology

Vol. 58, No. 8

Polynucleotide Sequence Relationships among Flagellin Genes of Campylobacterjejuni and Campylobacter coli SCOTT A. THORNTON,' SUSAN M. LOGAN,2 TREVOR J. TRUST,2 AND PATRICIA GUERRY1* Infectious Diseases Department, Naval Medical Research Institute, Bethesda, Maryland 20814,1 and Department of

Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2 Y2, Canada2 Received 7 February 1990/Accepted 24 April 1990

DNA probes that encode a complete flagellin gene and various internal regions of the Campylobacter coli VC167 flagellin genes were hybridized to 30 strains of C. coli or C. jejuni from 20 different Lior serogroups. The results indicated a high overall degree of homology among all of the strains examined. Although the most variable regions occurred within the middle of the gene, significant DNA homology was observed among many serogroups in this region of the molecule.

The thermophilic campylobacters Campylobacter jejuni and C. coli are significant causes of gastroenteritis worldwide (3, 4, 6, 23). There are considerable data implicating the flagella of these campylobacters in pathogenicity. Flagella are required for the organisms to colonize the mucous lining of an intestinal tract (17, 21, 22), and nonflagellated variants are unable to colonize animals (5) or human volunteers (3). Studies have shown that Campylobacter cells undergo both phase and antigenic variations of flagella. Phase variation refers to the abilities of some Campylobacter strains to express flagella reversibly (5); antigenic variation refers to the abilities of some strains to synthesize alternate flagellin subunits that are distinguishable antigenically and by apparent differences in Mr (9). In addition, flagella appear to be the major serodeterminant in certain serogroups of the Lior heat-labile serotyping scheme of campylobacters (9, 26), of which over 100 serogroups are currently recognized. The flagellin of campylobacters is the immunodominant antigen recognized during human and experimental animal infections, and studies with some animal models suggest that flagellin elicits some degree of protective immunity. This is based on findings obtained with animal models that indicate that previous exposure to one strain results in subsequent protection against strains of the same, but not a heterologous, Lior serotype (1, 2). In addition, passive protection with antiflagellar monoclonal antibodies has been demonstrated in a suckling mouse model (24). Despite the extensive antigenic diversity suggested by the Lior serotyping scheme, there is antigenic cross-reactivity among campylobacter flagellins (18). Campylobacter flagellin shows a high degree of sequence similarity to the flagellins of salmonellae and bacilli in the amino and carboxy ends of the molecule (19), which are known to be conserved among bacteria and which have been shown to function in transport and assembly of the monomers into the flagellar filament in enteric bacteria (10, 16). Presumably, as with Salmonella typhimurium and Escherichia coli (11, 12, 15, 16), the internal region of campylobacter flagellin contains antigenically variable sequences. These variable sequences most likely include the coding region for epitopes that are surface exposed in the intact flagellar filament and are responsible for serospecificity (19). To address the extent of the antigenic variability of campylobacter flagellins and to assess the practicality of developing subunit vaccines based on campylobacter flagel*

lin, we have constructed DNA probes from different regions of the flagellin genes of C. coli VC167 (7, 19) and used these probes to examine nucleotide sequence similarities among strains of different Lior serotypes by both colony and Southern blot hybridization analyses. C. coli VC167, which is the type strain of the L108 serogroup, has two full-length flagellin genes, flaA and flaB (7). The two genes are adjacent to one another in a tandem orientation on the chromosome and are 93% homologous at the nucleotide level (7). Figure 1 shows the restriction map of the flagellin gene cluster of C. coli VC167 and the regions of these genes used as probes. All of the plasmids used as probes contain only internal flagellin gene information, with the exception of pGK204. This plasmid contains a fulllength, circularly permuted flagellin gene; it contains the carboxy end of the flaA gene, the 163-base-pair intergenic region, and the amino-terminal end offlaB. Plasmid pGK203 contains the entire central region of the flaA gene, with 188 base pairs of the conserved amino-terminal end and 229 base pairs of the conserved carboxy-terminal end (19). Plasmids pGK206, pGK207, pGK208, and pGK209 contain various restriction fragments from the central region of the flaA gene. The Campylobacter strains used in this study are summarized in Table 1. For colony hybridization experiments, the cells were inoculated in a pattern onto an 82-mm-diameter nitrocellulose membrane filter overlying a campylobacter blood agar plate (Remel, Lenexa, Kans.). After overnight incubation at 37°C in a microaerophilic environment, the cells were lysed as described by Maniatis et al. (20) and baked. Southern blot hybridizations were performed as described by Maniatis et al. (20). Hybridizations to various flagellin probes which were labeled with [ot-32P]dCTP by nick translation (Dupont, NEN Research Products, Boston, Mass.) were performed under stringent conditions that have been previously described (8). The results of colony hybridization of the various probes against 30 campylobacter strains of 20 different Lior serotypes are summarized in Table 1. All of the strains hybridized strongly with pGK204, which was the only plasmid containing a complete flagellin gene. There was a gradation in the extent of hybridization with the other probes. Plasmid pGK203, which lacks most of the amino and carboxy regions, also hybridized to all of the strains, although the signal intensity of the colony hybridizations varied considerably (data not shown). Plasmid pGK206, which lacks 367 base

Corresponding author. 2686


VOL. 58, 1990


flaA H






100 bp



1 n

a I






pGK203 M








p0Ip209 FIG. 1. Flagellin gene probes. The top line depicts the organization of the two flagellin genes on the C. coli VC167 chromosome (7). The coding regions of flaA and flaB are indicated by open boxes; transcription of both genes is from left to right as drawn. Various restriction fragments were subcloned into pBR322 to generate the series of plasmids indicated. Restriction sites are marked as follows: H, HindlIl; R, EcoRI; RV, EcoRV; P, PstI; B, BctI. bp, Base pairs.

pairs of the leftward portion of the internal variable region, hybridized to most of the strains, but considerable mismatching was indicated in many of the serotypes. Strains of serogroups 2, 6, 7, 9, 13, 15, 20, 36, and 77 hybridized much more strongly than serotypes 1, 4, 5, 11, 12, 14, 17, 18, 32, and 46. Selected strains were examined by Southern blot

Lior serotype

8 8 8 77 77 77 77 77 77 77 7 15 13 20 6 36 2 9 32 4 1 5 11 11 12 14 46 46 17 18

analysis with plasmid pGK206 as the probe (Fig. 2A). Strains VC94 (LI05; lane 2), VC97 (LI020; lane 3), and VC84 (LI06; lane 4) hybridized as strongly as the homologous VC167 strain (LI08, lanes 1 and 9). Strains VC74 (LIO11; lane 5), VC208 (LIO 32; lane 6), VC212 (LI046; lane 7), and CL99 (LI046; lane 8) showed marked reduction in signal

TABLE 1. Hybridization of flagellin probes from VC167 to campylobacters of other serotypes Hybridization to probea: Strain Species pGK206 pGK207 pGK208 pGK203 pGK204 VC167 VC156 VC159 CL101 CL106 CL116 CL127 CL128 CL42 CL69 VC95 VC94

VC96 VC97 VC84 HC VC89 VC90 VC208 VC83 VC87 VC88 VC91 VC74 VC92 VC93 VC212 CL99 VC103 VC99

C. coli C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. coli C. jejuni C. coli C. coli C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. coli C. coli C. coli C. coli C. jejuni C. jejuni

+ + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + +

+ + +

+ + + + + + + + + + + + + + +


+ + + + + + + + + +

+ + + + + + + + + + +


_ _ _ _

_ + +

pGK209 + + +

+ +


_ + _ +


4++ + + + + + + + +


+ + +



a Hybridization to the various probes is summarized. +, Strong hybridization; ±, weak hybridization; +, no detectable hybridization. Hybridization conditions were as described by Guerry et al. (8). Each hybridization was repeated at least twice.







3 4
















*~~~~~~~~ _b








FIG. 2. Southern blot hybridizations of probes pGK206 (A) and pGK209 (B) to campylobacter DNAs. Lanes: 1, VC167 (LI08); 2, VC94 (LIO5); 3, VC97 (LI020); 4, VC84 (LI06); 5, VC74 (LIO11); 6, VC208 (LI032); 7, VC212 (LI046); 8, CL99 (LI046); 9, VC167 (LI08). DNAs were

extracted as previously described (8). pBR322 vector sequences do not hybridize to campylobacter DNA under the conditions used (8).

strength, although they clearly share some sequences in multiple flagellin genes strains (19). Plasmids pGK207, pGK208, and pGK209 showed decreasing specificities by colony blots (Table 1). Although plasmid pGK209 seemed to hybridize only to LI08 and L1077 strains by colony blots, Southern analysis demonstrated a weak hybridization signal to multiple serotypes (Fig. 2B). However, the hybridization signal to serotypes L105 (lane 2), L1020 (lane 3), and LI06 (lane 4) was also stronger than the signal seen with LIOll (lane 5), LI032 (lane 6), and LI046 (lanes 7 and 8). The data confirm that the amino and carboxy ends of campylobacter flagellins are conserved and that there is considerable heterogeneity in the internal regions of the molecule. However, the data also suggest that campylobacter flagellins are more conserved than the Lior serotyping scheme indicates. Although we examined a limited number of strains, the data showed considerable sequence similarities among the internal regions of 9 serotypes (LI02, L106, LI07, LI08, L1013, LIO15, L1020, L1036, and L1077) and more limited homology of the internal region of L108 to the internal regions of 11 other serotypes (LIO1, L104, LIO5, L109, LIO11, LI012, LI014, L1017, L1018, L1032, and L1046). This suggests that some serological distinctions may be based on relatively minor changes, as with S. typhimurium (13, 14, 25). Work is ongoing in our laboratories to identify surface-exposed epitopes from VC167 flagellin and assess their vaccine potential against homologous and heterologous serogroups. If some of the conserved sequences are surface exposed, it is possible that the serospecific protective immunity reported by some workers (1, 2, 24) extends to a limited number of related serogroups. common. This blot also suggests that are found in many campylobacter

This work was supported in part by U.S. Navy Research and Development Command Research Work Unit 61102A3M161102 BS13 AK.111 and by a grant from the Medical Research Council of Canada to T.J.T. We thank Hermy Lior for serotyping and Steve Martin for expert technical assistance. LITERATURE CITED

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VOL. 58, 1990 19. Logan, S. M., T. J. Trust, and P. Guerry. 1989. Evidence for posttranslational modification and gene duplication of Campylobacter flagellin. J. Bacteriol. 171:3031-3038. 20. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Morooka, T., A. Umeda, and K. Amako. 1985. Motility as an intestinal colonization factor for Campylobacterjejuni. J. Gen. Microbiol. 131:1973-1980. 22. Newell, D. G. 1986. Monoclonal antibodies directed against the flagella of Campylobacter jejuni: production, characterization, and lack of effect on the colonization of infant mice. J. Hyg. 96:131-141.



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Polynucleotide sequence relationships among flagellin genes of Campylobacter jejuni and Campylobacter coli.

DNA probes that encode a complete flagellin gene and various internal regions of the Campylobacter coli VC167 flagellin genes were hybridized to 30 st...
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