Exchange of Lipooligosaccharide Synthesis Genes Creates Potential Guillain-Barre Syndrome-Inducible Strains of Campylobacter jejuni
http://www.100md.com
感染与免疫杂志 2006年第2期
Biotechnology and Environmental Biology, School of Applied Sciences, Royal Melbourne Institute of Technology University, Melbourne, Victoria, Australia
ABSTRACT
Human ganglioside-like structures, such as GM1, found on some Campylobacter jejuni strains have been linked to inducing the Guillain-Barre Syndrome (GBS). This study shows that a C. jejuni strain without GM1-like molecules acquired large DNA fragments, including lipooligosaccharide synthesis genes, from a strain expressing GM1-like molecules and consequently transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity.
TEXT
The Guillain-Barre Syndrome (GBS) is a postinfectious autoimmune neuropathy that can occur following campylobacteriosis. Affected persons rapidly develop weakness of the limbs, weakness of the respiratory muscles, and areflexia (11). Presently, the pathogenesis of GBS is not fully understood. However, since similar C. jejuni isolates have been isolated from both GBS and non-GBS patients (17), host factors also play an important role in GBS development. In addition, a study by Yuki et al. showed that rabbits, which had been sensitized with C. jejuni lipooligosaccharide (LOS), developed anti-ganglioside-GM1 immunoglobulin G (IgG) antibodies and flaccid limb weakness (18). Paralyzed rabbits had pathological changes in their peripheral nerves that were identical to those present in GBS (18). Moreover, immunization of mice with C. jejuni LOS generated a monoclonal antibody (MAb) that reacted with GM1 and bound to human peripheral nerves. The MAb and anti-GM1 IgG from GBS patients blocked muscle action potentials in a muscle-spinal cord coculture (18). These results indicate that anti-GM1 antibodies can cause muscle weakness (18), and the molecular mimicry that exists between the human gangliosides, including GM1, and the C. jejuni LOS is one of the GBS-inducible determinants.
The C. jejuni LOS is partly encoded by the wlaII gene cluster that has been shown to exhibit a high degree of variation among strains (6, 14) (Fig. 1). Presently, the function of individual LOS genes is not fully understood; however, the wlaND, cgtA, cgtB, cstII, neuB, neuC, neuA, and waaF genes are essential for the formation of human ganglioside-like LOS structures which can induce GBS (7-10, 12, 18). Upstream of the waaC gene, the wlaI gene cluster is found which is highly conserved in this bacterium (4, 15) (Fig. 1). The wlaI locus is mainly involved in protein glycosylation, although at least one gene, galE, is also involved in LOS synthesis (3).
Natural transformation is the ability of a bacterium to take up genetic material, which can be integrated into the chromosomal DNA via homologous recombination. Exchange of LOS synthesis genes was recently shown for a C. jejuni strain isolated from a patient with GBS (5). In the present study, we hypothesized that a non-GM1 strain could take up LOS synthesis genes in vitro and become a GBS-inducible strain. To test this hypothesis, C. jejuni strain 81116 (Penner serotype O:6, named WT 81116), which was originally isolated from a human waterborne outbreak of gastroenteritis (13), was selected as the host cell in a natural transformation experiment. The LOS of this strain does not react with the cholera toxin B subunit (CTB), which is GM1 specific. C. jejuni Penner serotype O:4 (named WT O:4) was selected as the donor cell because (i) it strongly reacted with CTB, (ii) the C. jejuni O:4 wlaVA mutant (named O:4 wlaVA) could be successfully constructed by introducing the selection marker for kanamycin resistance and still reacted with CTB, and (iii) we previously showed that mutating the wlaVA gene in a strain HB 93-13 (O:19) did not affect the LOS structure, the LOS sugar composition, and the ganglioside mimicry (unpublished data). C. jejuni 81116 was grown on 5% defribinated horse blood-supplemented Columbia agar at 37°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2) for 16 h. The bacterial cells were harvested and suspended in 1 ml of heart infusion broth, and 50 μl of bacterial suspension was preincubated in an Eppendorf tube (1.5 ml in size) containing 1 ml of heart infusion agar under microaerobic conditions for 2 h. Subsequently, 10 μg of isolated chromosomal DNA of O:4 wlaVA was added, and the mixture was incubated under the same conditions for 3 h. After incubation, the bacteria were grown as previously mentioned on selective medium supplemented with kanamycin (50 μg/ml) for 48 h. The transformation efficiency of WT 81116 was approximately 2 x 102 CFU per 10 μg of genomic DNA. One hundred and fifty kanamycin resistance colonies were randomly picked and immunologically probed with CTB to screen for GM1-positive transformants. Surprisingly, 145 of 150 colonies reacted with CTB, whereas only 5 colonies were CTB negative (Fig. 2, selected transformants). Therefore, these results showed that C. jejuni 81116 could be transformed into a number of potential GBS-inducible transformants.
To identify which O:4 wlaVA genes had been acquired by the CTB-positive transformants, PCR-restriction fragment length polymorphism (PCR-RFLP) with HindIII as the restriction enzyme was performed. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer WaaC-F (5'-CCGTGGTTTTGCAATTTATC-3'), which is located in the waaC gene (nucleotides 53 to 72), and the primer WaaF-R (5'-AAGTTCTTGTTCGGCTTTTC-3'), which is located in the waaF gene (nucleotides 594 to 575), were designed. These primers were used to amplify the entire wlaII gene cluster of WT O:4 (13.558 kb), O:4 wlaVA (14.055 kb), WT 81116 (16.308 kb), 13 selected CTB-positive transformants (B, C, D, E, F, G, H, I, J, K, N, P, and R), and all 5 CTB-negative transformants (A, L, M, O, and Q). PCR was carried out by using the Expand Long Template PCR system (Roche). For the CTB-positive transformants, the entire wlaII gene cluster (16.308 kb) was replaced by the wlaII locus of O:4 wlaVA (14.095 kb) as the PCR-RFLP patterns of these transformants were identical to the pattern of O:4 wlaVA and markedly different from that of WT 81116. For the CTB-negative transformants L, M, O, and Q, a partial exchange of LOS synthesis genes was observed. Presumably, some essential gene(s) for the synthesis of the GM1-like LOS and, hence, CTB binding is missing from their wlaII gene clusters. Surprisingly, CTB-negative transformant A had received the complete wlaII locus of O:4 wlaVA (Fig. 3, see also Fig. 2). A possible explanation could be a change in the length of some of the homopolymeric tracts found in this gene cluster or mutations in the LOS synthesis genes (9) or other genes that are essential in the expression of the CTB-binding epitope. These results showed that part and even the entire wlaII gene cluster was easily taken up and integrated in the genome of C. jejuni 81116, resulting in new strains carrying GM1-like LOS structures.
To identify the integration point upstream of the waaC gene, HhaI-PCR-RFLP was performed as previously mentioned. Using the sequence data of the wlaI gene cluster of C. jejuni 81116 (4), the primer GalE1 (5'-GCGGTGGTGCAGGTTATATAGG-3'), which is located in the galE gene (nucleotides 17 to 38), and the primer WlaM (5'-GCTCACTCCACCGATAAGAT-3'), which is located in the wlaM gene (nucleotides 831 to 812), were designed. These primers were used to amplify the wlaI gene cluster of WT 81116 (14.061 kb), 81116 transformants (A to J), WT O:4, and O:4 wlaVA. The PCR products of approximately 14 kb were obtained for all strains. PCR-RFLP patterns of transformants showed a high degree of variation within the wlaI gene clusters, and those were different from the patterns of WT 81116 and O:4 wlaVA (Fig. 4). Restriction mapping analysis showed that WT 81116 randomly integrated DNA fragments from the O:4 wlaVA into its genome using various integration points. Most of them were positioned in the region of the galE gene as a 698-bp DNA fragment containing the partial galE gene was missing from the wlaI loci of transformants A, B, C, D, F, H, I, and J. A previous study in a C. jejuni strain GB11 also evidenced the integration point in the galE region (5). Other sites were distributed throughout the wlaI gene cluster such as in the wlaK, wlaF, wlaE, and wlaC genes.
To identify the integration point downstream of the waaF gene, HindIII-PCR-RFLP was performed. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer WaaF-F (5'-TACATCTTCCCACCTGGTTA-3'), which is located in the waaF gene (nucleotides 14 to 33), and the primer Cj1155c-R (5'-ATGCTTGCACCATACCTTTG-3'), which is located in the Cj1155c gene (nucleotides 116 to 97), were designed. These primers were used to amplify a 5.102-kb DNA fragment ranging from waaF to Cj1155c. PCR was carried out by using the Pfu polymerase (Roche). A PCR product of approximately 7 kb was obtained for the WT 81116, while a 5.1-kb PCR product was obtained for WT O:4, O:4 wlaVA, and transformants B, C, D, E, F, G, H, and J. A PCR product was not amplified from transformants A and I. RFLP analysis of PCR products showed that all transformants tested had the pattern of the donor DNA, and the site of recombination appears to be located in or downstream of the Cj1155c homologous region.
To identify the integration point downstream of the Cj1155c homologous region, HindIII-PCR-RFLP was performed as described above. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer Cj1155c-F (5'-AGGTATGGTGCAAGCATTAT-3'), which is located in the Cj1155c gene (nucleotides 100 to 119), and the primer DnaX-R (5'-TAGGCTCTCCAAAACAATCT-3'), which is located in the dnaX gene (nucleotides 1516 to 1497), were designed. These primers were used to amplify a 5.185-bp DNA fragment ranging from Cj1155c to dnaX. A PCR product of approximately 5.2 kb was obtained for WT O:4, O:4 wlaVA, WT 81116, and 81116 transformants (B to J). A PCR product was not amplified from transformants A. The integration point for transformants B, D, E, F, and H was located in or downstream of the dnaX homologous region since their RFLP patterns were identical to the RFLP pattern of O:4 wlaVA. For transformants C, G, I, and J, the integration point was located in the Cj1155c homologous region because their RFLP patterns were identical to the pattern of WT 81116. The most upstream and downstream integration points for each 81116 transformant and the approximate sizes of WT 81116 DNA that were deleted during genetic recombination were conclusively shown (Fig. 1).
To determine whether natural transformation with chromosomal DNA resulted in new genotypes of C. jejuni, pulsed-field gel electrophoresis (PFGE) with SacII as the restriction enzyme was performed. It was found that the SacII-PFGE patterns of the transformants A, B, C, D, E, F, H, I, and J were identical to the PFGE pattern of WT 81116 (Fig. 5, see representative PFGE pattern of transformant A). Interestingly, the PFGE pattern of the transformant G was different from that of WT 81116. It seems a large 388-kb DNA fragment and a 135-kb fragment of the O:4 wlaVA were inserted into the chromosomal DNA of WT 81116. Presumably, this insertion is at a location other than the LOS gene cluster since the change involving the LOS genes is apparently too small to be detected by PFGE. This indicated that not only the LOS gene cluster but also other locations throughout the WT 81116 genome had undergone a genetic exchange. These results showed that natural transformation can result in genotypic diversity, as also shown by previous studies (1, 2, 16).
To examine the LOS molecules of transformants A to J, L, M, O, and Q, LOS was isolated and separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. The LOS patterns of all transformants were different from the LOS patterns of both WT 81116 and O:4 wlaVA, except for LOS isolated from transformant Q that showed a similar LOS pattern to the WT 81116. Interestingly, the CTB-positive transformants (B, C, D, E, F, G, H, I, and J) carrying the LOS gene cluster of O:4 wlaVA showed a different LOS pattern to that of O:4 wlaVA. The cause for this difference is unclear and requires further investigation. As expected, the LOS patterns of WT O:4 and O:4 wlaVA were similar (Fig. 6). These results indicated that not only the wlaII gene cluster but also other genes are involved in LOS synthesis.
In conclusion, it was shown that a non-GM1 C. jejuni strain can take up DNA in vitro and transform into a number of potential GBS-inducing strains. Furthermore, we have shown that horizontal gene transfer between C. jejuni strains can result in genome plasticity. This result directly limits the current typing systems and complicates epidemiological studies. Moreover, since the incidence of mixed infections in areas of endemicity is high and could lead to the acquisition of virulence genes by nonpathogenic strains via interstrain genetic exchange, this result also indicates potential risks in using C. jejuni as a live vaccine in both animals and humans.
REFERENCES
1. Alm, R. A., P. Guerry, and T. J. Trust. 1993. Significance of duplicated flagellin genes in Campylobacter. J. Mol. Biol. 230:359-363.
2. Boer, P., J. A. Wagenaar, R. P. Achterberg, J. P. Putten, L. M. Schouls, and B. Duim. 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44:351-359.
3. Fry, B. N., S. Feng, Y. Y. Chen, D. G. Newell, P. J. Coloe, and V. Korolik. 2000. The galE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence. Infect. Immun. 68:2594-2601.
4. Fry, B. N., V. Korolik, J. A. ten Brinke, M. T. Pennings, R. Zalm, B. J. Teunis, P. J. Coloe, and B. A. van der Zeijst. 1998. The lipopolysaccharide biosynthesis locus of Campylobacter jejuni 81116. Microbiology 144(Pt. 8):2049-2061.
5. Gilbert, M., P. C. Godschalk, M. F. Karwaski, C. W. Ang, A. van Belkum, J. Li, W. W. Wakarchuk, and H. P. Endtz. 2004. Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barre syndrome, by horizontal exchange. Infect. Immun. 72:1162-1165.
6. Gilbert, M., M. F. Karwaski, S. Bernatchez, N. M. Young, E. Taboada, J. Michniewicz, A. M. Cunningham, and W. W. Wakarchuk. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277:327-337.
7. Guerry, P., C. P. Ewing, T. E. Hickey, M. M. Prendergast, and A. P. Moran. 2000. Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni. Infect. Immun. 68:6656-6662.
8. Guerry, P., C. M. Szymanski, M. M. Prendergast, T. E. Hickey, C. P. Ewing, D. L. Pattarini, and A. P. Moran. 2002. Phase variation of Campylobacter jejuni 81-176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro. Infect. Immun. 70:787-793.
9. Linton, D., M. Gilbert, P. G. Hitchen, A. Dell, H. R. Morris, W. W. Wakarchuk, N. A. Gregson, and B. W. Wren. 2000. Phase variation of a -1,3-galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni. Mol. Microbiol. 37:501-514.
10. Linton, D., A. V. Karlyshev, P. G. Hitchen, H. R. Morris, A. Dell, N. A. Gregson, and B. W. Wren. 2000. Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide. Mol. Microbiol. 35:1120-1134.
11. Nachamkin, I., B. M. Allos, and T. Ho. 1998. Campylobacter species and Guillain-Barre syndrome. Clin. Microbiol. Rev. 11:555-567.
12. Oldfield, N. J., A. P. Moran, L. A. Millar, M. M. Prendergast, and J. M. Ketley. 2002. Characterization of the Campylobacter jejuni heptosyltransferase II gene, waaF, provides genetic evidence that extracellular polysaccharide is lipid A core independent. J. Bacteriol. 184:2100-2107.
13. Palmer, S. R., P. R. Gully, J. M. White, A. D. Pearson, W. G. Suckling, D. M. Jones, J. C. Rawes, and J. L. Penner. 1983. Water-borne outbreak of Campylobacter gastroenteritis. Lancet i:287-290.
14. Parker, C. T., S. T. Horn, M. Gilbert, W. G. Miller, D. L. Woodward, and R. E. Mandrell. 2005. Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources. J. Clin. Microbiol. 43:2771-2781.
15. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev, S. Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H. van Vliet, S. Whitehead, and B. G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668.
16. Wassenaar, T. M., B. N. Fry, and B. A. van der Zeijst. 1995. Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer. Microbiology 141(Pt. 1):95-101.
17. Watanabe, H., K. Shindo, Y. Nakamura, M. Nagamatsu, Z. Shiozawa, and S. Kusunoki. 2001. A case of Guillain-Barre syndrome after Campylobacter jejuni enterocolitis: anti-ganglioside antibody levels with or without Guillain-Barre syndrome. Rinsho Shinkeigaku 41:625-627.
18. Yuki, N., K. Susuki, M. Koga, Y. Nishimoto, M. Odaka, K. Hirata, K. Taguchi, T. Miyatake, K. Furukawa, T. Kobata, and M. Yamada. 2004. Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain-Barre syndrome. Proc. Natl. Acad. Sci. USA 101:11404-11409.(Vongsavanh Phongsisay, Vi)
ABSTRACT
Human ganglioside-like structures, such as GM1, found on some Campylobacter jejuni strains have been linked to inducing the Guillain-Barre Syndrome (GBS). This study shows that a C. jejuni strain without GM1-like molecules acquired large DNA fragments, including lipooligosaccharide synthesis genes, from a strain expressing GM1-like molecules and consequently transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity.
TEXT
The Guillain-Barre Syndrome (GBS) is a postinfectious autoimmune neuropathy that can occur following campylobacteriosis. Affected persons rapidly develop weakness of the limbs, weakness of the respiratory muscles, and areflexia (11). Presently, the pathogenesis of GBS is not fully understood. However, since similar C. jejuni isolates have been isolated from both GBS and non-GBS patients (17), host factors also play an important role in GBS development. In addition, a study by Yuki et al. showed that rabbits, which had been sensitized with C. jejuni lipooligosaccharide (LOS), developed anti-ganglioside-GM1 immunoglobulin G (IgG) antibodies and flaccid limb weakness (18). Paralyzed rabbits had pathological changes in their peripheral nerves that were identical to those present in GBS (18). Moreover, immunization of mice with C. jejuni LOS generated a monoclonal antibody (MAb) that reacted with GM1 and bound to human peripheral nerves. The MAb and anti-GM1 IgG from GBS patients blocked muscle action potentials in a muscle-spinal cord coculture (18). These results indicate that anti-GM1 antibodies can cause muscle weakness (18), and the molecular mimicry that exists between the human gangliosides, including GM1, and the C. jejuni LOS is one of the GBS-inducible determinants.
The C. jejuni LOS is partly encoded by the wlaII gene cluster that has been shown to exhibit a high degree of variation among strains (6, 14) (Fig. 1). Presently, the function of individual LOS genes is not fully understood; however, the wlaND, cgtA, cgtB, cstII, neuB, neuC, neuA, and waaF genes are essential for the formation of human ganglioside-like LOS structures which can induce GBS (7-10, 12, 18). Upstream of the waaC gene, the wlaI gene cluster is found which is highly conserved in this bacterium (4, 15) (Fig. 1). The wlaI locus is mainly involved in protein glycosylation, although at least one gene, galE, is also involved in LOS synthesis (3).
Natural transformation is the ability of a bacterium to take up genetic material, which can be integrated into the chromosomal DNA via homologous recombination. Exchange of LOS synthesis genes was recently shown for a C. jejuni strain isolated from a patient with GBS (5). In the present study, we hypothesized that a non-GM1 strain could take up LOS synthesis genes in vitro and become a GBS-inducible strain. To test this hypothesis, C. jejuni strain 81116 (Penner serotype O:6, named WT 81116), which was originally isolated from a human waterborne outbreak of gastroenteritis (13), was selected as the host cell in a natural transformation experiment. The LOS of this strain does not react with the cholera toxin B subunit (CTB), which is GM1 specific. C. jejuni Penner serotype O:4 (named WT O:4) was selected as the donor cell because (i) it strongly reacted with CTB, (ii) the C. jejuni O:4 wlaVA mutant (named O:4 wlaVA) could be successfully constructed by introducing the selection marker for kanamycin resistance and still reacted with CTB, and (iii) we previously showed that mutating the wlaVA gene in a strain HB 93-13 (O:19) did not affect the LOS structure, the LOS sugar composition, and the ganglioside mimicry (unpublished data). C. jejuni 81116 was grown on 5% defribinated horse blood-supplemented Columbia agar at 37°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2) for 16 h. The bacterial cells were harvested and suspended in 1 ml of heart infusion broth, and 50 μl of bacterial suspension was preincubated in an Eppendorf tube (1.5 ml in size) containing 1 ml of heart infusion agar under microaerobic conditions for 2 h. Subsequently, 10 μg of isolated chromosomal DNA of O:4 wlaVA was added, and the mixture was incubated under the same conditions for 3 h. After incubation, the bacteria were grown as previously mentioned on selective medium supplemented with kanamycin (50 μg/ml) for 48 h. The transformation efficiency of WT 81116 was approximately 2 x 102 CFU per 10 μg of genomic DNA. One hundred and fifty kanamycin resistance colonies were randomly picked and immunologically probed with CTB to screen for GM1-positive transformants. Surprisingly, 145 of 150 colonies reacted with CTB, whereas only 5 colonies were CTB negative (Fig. 2, selected transformants). Therefore, these results showed that C. jejuni 81116 could be transformed into a number of potential GBS-inducible transformants.
To identify which O:4 wlaVA genes had been acquired by the CTB-positive transformants, PCR-restriction fragment length polymorphism (PCR-RFLP) with HindIII as the restriction enzyme was performed. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer WaaC-F (5'-CCGTGGTTTTGCAATTTATC-3'), which is located in the waaC gene (nucleotides 53 to 72), and the primer WaaF-R (5'-AAGTTCTTGTTCGGCTTTTC-3'), which is located in the waaF gene (nucleotides 594 to 575), were designed. These primers were used to amplify the entire wlaII gene cluster of WT O:4 (13.558 kb), O:4 wlaVA (14.055 kb), WT 81116 (16.308 kb), 13 selected CTB-positive transformants (B, C, D, E, F, G, H, I, J, K, N, P, and R), and all 5 CTB-negative transformants (A, L, M, O, and Q). PCR was carried out by using the Expand Long Template PCR system (Roche). For the CTB-positive transformants, the entire wlaII gene cluster (16.308 kb) was replaced by the wlaII locus of O:4 wlaVA (14.095 kb) as the PCR-RFLP patterns of these transformants were identical to the pattern of O:4 wlaVA and markedly different from that of WT 81116. For the CTB-negative transformants L, M, O, and Q, a partial exchange of LOS synthesis genes was observed. Presumably, some essential gene(s) for the synthesis of the GM1-like LOS and, hence, CTB binding is missing from their wlaII gene clusters. Surprisingly, CTB-negative transformant A had received the complete wlaII locus of O:4 wlaVA (Fig. 3, see also Fig. 2). A possible explanation could be a change in the length of some of the homopolymeric tracts found in this gene cluster or mutations in the LOS synthesis genes (9) or other genes that are essential in the expression of the CTB-binding epitope. These results showed that part and even the entire wlaII gene cluster was easily taken up and integrated in the genome of C. jejuni 81116, resulting in new strains carrying GM1-like LOS structures.
To identify the integration point upstream of the waaC gene, HhaI-PCR-RFLP was performed as previously mentioned. Using the sequence data of the wlaI gene cluster of C. jejuni 81116 (4), the primer GalE1 (5'-GCGGTGGTGCAGGTTATATAGG-3'), which is located in the galE gene (nucleotides 17 to 38), and the primer WlaM (5'-GCTCACTCCACCGATAAGAT-3'), which is located in the wlaM gene (nucleotides 831 to 812), were designed. These primers were used to amplify the wlaI gene cluster of WT 81116 (14.061 kb), 81116 transformants (A to J), WT O:4, and O:4 wlaVA. The PCR products of approximately 14 kb were obtained for all strains. PCR-RFLP patterns of transformants showed a high degree of variation within the wlaI gene clusters, and those were different from the patterns of WT 81116 and O:4 wlaVA (Fig. 4). Restriction mapping analysis showed that WT 81116 randomly integrated DNA fragments from the O:4 wlaVA into its genome using various integration points. Most of them were positioned in the region of the galE gene as a 698-bp DNA fragment containing the partial galE gene was missing from the wlaI loci of transformants A, B, C, D, F, H, I, and J. A previous study in a C. jejuni strain GB11 also evidenced the integration point in the galE region (5). Other sites were distributed throughout the wlaI gene cluster such as in the wlaK, wlaF, wlaE, and wlaC genes.
To identify the integration point downstream of the waaF gene, HindIII-PCR-RFLP was performed. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer WaaF-F (5'-TACATCTTCCCACCTGGTTA-3'), which is located in the waaF gene (nucleotides 14 to 33), and the primer Cj1155c-R (5'-ATGCTTGCACCATACCTTTG-3'), which is located in the Cj1155c gene (nucleotides 116 to 97), were designed. These primers were used to amplify a 5.102-kb DNA fragment ranging from waaF to Cj1155c. PCR was carried out by using the Pfu polymerase (Roche). A PCR product of approximately 7 kb was obtained for the WT 81116, while a 5.1-kb PCR product was obtained for WT O:4, O:4 wlaVA, and transformants B, C, D, E, F, G, H, and J. A PCR product was not amplified from transformants A and I. RFLP analysis of PCR products showed that all transformants tested had the pattern of the donor DNA, and the site of recombination appears to be located in or downstream of the Cj1155c homologous region.
To identify the integration point downstream of the Cj1155c homologous region, HindIII-PCR-RFLP was performed as described above. Using the genome sequence of C. jejuni NCTC 11168 (15), the primer Cj1155c-F (5'-AGGTATGGTGCAAGCATTAT-3'), which is located in the Cj1155c gene (nucleotides 100 to 119), and the primer DnaX-R (5'-TAGGCTCTCCAAAACAATCT-3'), which is located in the dnaX gene (nucleotides 1516 to 1497), were designed. These primers were used to amplify a 5.185-bp DNA fragment ranging from Cj1155c to dnaX. A PCR product of approximately 5.2 kb was obtained for WT O:4, O:4 wlaVA, WT 81116, and 81116 transformants (B to J). A PCR product was not amplified from transformants A. The integration point for transformants B, D, E, F, and H was located in or downstream of the dnaX homologous region since their RFLP patterns were identical to the RFLP pattern of O:4 wlaVA. For transformants C, G, I, and J, the integration point was located in the Cj1155c homologous region because their RFLP patterns were identical to the pattern of WT 81116. The most upstream and downstream integration points for each 81116 transformant and the approximate sizes of WT 81116 DNA that were deleted during genetic recombination were conclusively shown (Fig. 1).
To determine whether natural transformation with chromosomal DNA resulted in new genotypes of C. jejuni, pulsed-field gel electrophoresis (PFGE) with SacII as the restriction enzyme was performed. It was found that the SacII-PFGE patterns of the transformants A, B, C, D, E, F, H, I, and J were identical to the PFGE pattern of WT 81116 (Fig. 5, see representative PFGE pattern of transformant A). Interestingly, the PFGE pattern of the transformant G was different from that of WT 81116. It seems a large 388-kb DNA fragment and a 135-kb fragment of the O:4 wlaVA were inserted into the chromosomal DNA of WT 81116. Presumably, this insertion is at a location other than the LOS gene cluster since the change involving the LOS genes is apparently too small to be detected by PFGE. This indicated that not only the LOS gene cluster but also other locations throughout the WT 81116 genome had undergone a genetic exchange. These results showed that natural transformation can result in genotypic diversity, as also shown by previous studies (1, 2, 16).
To examine the LOS molecules of transformants A to J, L, M, O, and Q, LOS was isolated and separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. The LOS patterns of all transformants were different from the LOS patterns of both WT 81116 and O:4 wlaVA, except for LOS isolated from transformant Q that showed a similar LOS pattern to the WT 81116. Interestingly, the CTB-positive transformants (B, C, D, E, F, G, H, I, and J) carrying the LOS gene cluster of O:4 wlaVA showed a different LOS pattern to that of O:4 wlaVA. The cause for this difference is unclear and requires further investigation. As expected, the LOS patterns of WT O:4 and O:4 wlaVA were similar (Fig. 6). These results indicated that not only the wlaII gene cluster but also other genes are involved in LOS synthesis.
In conclusion, it was shown that a non-GM1 C. jejuni strain can take up DNA in vitro and transform into a number of potential GBS-inducing strains. Furthermore, we have shown that horizontal gene transfer between C. jejuni strains can result in genome plasticity. This result directly limits the current typing systems and complicates epidemiological studies. Moreover, since the incidence of mixed infections in areas of endemicity is high and could lead to the acquisition of virulence genes by nonpathogenic strains via interstrain genetic exchange, this result also indicates potential risks in using C. jejuni as a live vaccine in both animals and humans.
REFERENCES
1. Alm, R. A., P. Guerry, and T. J. Trust. 1993. Significance of duplicated flagellin genes in Campylobacter. J. Mol. Biol. 230:359-363.
2. Boer, P., J. A. Wagenaar, R. P. Achterberg, J. P. Putten, L. M. Schouls, and B. Duim. 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44:351-359.
3. Fry, B. N., S. Feng, Y. Y. Chen, D. G. Newell, P. J. Coloe, and V. Korolik. 2000. The galE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence. Infect. Immun. 68:2594-2601.
4. Fry, B. N., V. Korolik, J. A. ten Brinke, M. T. Pennings, R. Zalm, B. J. Teunis, P. J. Coloe, and B. A. van der Zeijst. 1998. The lipopolysaccharide biosynthesis locus of Campylobacter jejuni 81116. Microbiology 144(Pt. 8):2049-2061.
5. Gilbert, M., P. C. Godschalk, M. F. Karwaski, C. W. Ang, A. van Belkum, J. Li, W. W. Wakarchuk, and H. P. Endtz. 2004. Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barre syndrome, by horizontal exchange. Infect. Immun. 72:1162-1165.
6. Gilbert, M., M. F. Karwaski, S. Bernatchez, N. M. Young, E. Taboada, J. Michniewicz, A. M. Cunningham, and W. W. Wakarchuk. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277:327-337.
7. Guerry, P., C. P. Ewing, T. E. Hickey, M. M. Prendergast, and A. P. Moran. 2000. Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni. Infect. Immun. 68:6656-6662.
8. Guerry, P., C. M. Szymanski, M. M. Prendergast, T. E. Hickey, C. P. Ewing, D. L. Pattarini, and A. P. Moran. 2002. Phase variation of Campylobacter jejuni 81-176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro. Infect. Immun. 70:787-793.
9. Linton, D., M. Gilbert, P. G. Hitchen, A. Dell, H. R. Morris, W. W. Wakarchuk, N. A. Gregson, and B. W. Wren. 2000. Phase variation of a -1,3-galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni. Mol. Microbiol. 37:501-514.
10. Linton, D., A. V. Karlyshev, P. G. Hitchen, H. R. Morris, A. Dell, N. A. Gregson, and B. W. Wren. 2000. Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide. Mol. Microbiol. 35:1120-1134.
11. Nachamkin, I., B. M. Allos, and T. Ho. 1998. Campylobacter species and Guillain-Barre syndrome. Clin. Microbiol. Rev. 11:555-567.
12. Oldfield, N. J., A. P. Moran, L. A. Millar, M. M. Prendergast, and J. M. Ketley. 2002. Characterization of the Campylobacter jejuni heptosyltransferase II gene, waaF, provides genetic evidence that extracellular polysaccharide is lipid A core independent. J. Bacteriol. 184:2100-2107.
13. Palmer, S. R., P. R. Gully, J. M. White, A. D. Pearson, W. G. Suckling, D. M. Jones, J. C. Rawes, and J. L. Penner. 1983. Water-borne outbreak of Campylobacter gastroenteritis. Lancet i:287-290.
14. Parker, C. T., S. T. Horn, M. Gilbert, W. G. Miller, D. L. Woodward, and R. E. Mandrell. 2005. Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources. J. Clin. Microbiol. 43:2771-2781.
15. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev, S. Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H. van Vliet, S. Whitehead, and B. G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668.
16. Wassenaar, T. M., B. N. Fry, and B. A. van der Zeijst. 1995. Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer. Microbiology 141(Pt. 1):95-101.
17. Watanabe, H., K. Shindo, Y. Nakamura, M. Nagamatsu, Z. Shiozawa, and S. Kusunoki. 2001. A case of Guillain-Barre syndrome after Campylobacter jejuni enterocolitis: anti-ganglioside antibody levels with or without Guillain-Barre syndrome. Rinsho Shinkeigaku 41:625-627.
18. Yuki, N., K. Susuki, M. Koga, Y. Nishimoto, M. Odaka, K. Hirata, K. Taguchi, T. Miyatake, K. Furukawa, T. Kobata, and M. Yamada. 2004. Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain-Barre syndrome. Proc. Natl. Acad. Sci. USA 101:11404-11409.(Vongsavanh Phongsisay, Vi)