INFECTION AND IMMUNITY, Feb. 2004, p. 1162–1165 0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.2.1162–1165.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 2
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 Michel Gilbert,1* Peggy C. R. Godschalk,2 Marie-France Karwaski,1 C. Wim Ang,2 Alex van Belkum,2 Jianjun Li,1 Warren W. Wakarchuk,1 and Hubert P. Endtz2 Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada,1 and Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands2 Received 8 July 2003/Returned for modification 4 September 2003/Accepted 12 November 2003
Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barre´ syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168. We sequenced the LOS biosynthesis locus of GB11 and found that it was nearly identical to the class A LOS locus from the C. jejuni HS:19 Penner serotype strain (ATCC 43446). Analysis of the DNA sequencing data showed that a horizontal exchange event involving at least 14.26 kb had occurred in the LOS biosynthesis locus of GB11 between galE (Cj1131c in NCTC 11168) and gmhA (Cj1149 in NCTC 11168). Mass spectrometry of the GB11 LOS showed that GB11 expressed an LOS outer core that mimicked the carbohydrate portion of the gangliosides GM1a and GD1a, similar to C. jejuni ATCC 43446. The serum from the GB11-infected patient was shown to react with the LOS from both GB11 and ATCC 43446 but not with that from NCTC 11168. These data indicate that the antiganglioside response in the GB11-infected patient was raised against the structures synthesized by the acquired class A LOS locus. GBS and MFS strains were compared with enteritis-only strains, C. jejuni GB11, a strain isolated from a patient with GBS, was found in the same cluster as C. jejuni NCTC 11168 (the “genome strain”). C. jejuni GB11 and NCTC 11168 have the same HS (HS:2) and heat-labile (Lior type 4) serotypes. Other evidence that C. jejuni GB11 is genetically close to NCTC 11168 is provided by MLST of seven housekeeping genes and of the flaA short variable region (SVR). With the use of MLST, both C. jejuni NCTC 11168 and GB11 were shown to belong to the ST-21 clonal complex (6; http://campylobacter.mlst.net). Also, the sequences of the flaA SVRs of C. jejuni NCTC 11168 (GenBank accession number AL139078) and GB11 (GenBank accession number AF354548) are 100% (321 of 321 bp) identical. Because C. jejuni GB11 is genetically related to the genome strain NCTC 11168, it would be a good candidate for a GBS prototype strain to identify any potential neuropathogenic factor specific to GBS and MFS strains. However, there is no way to prove that C. jejuni NCTC 11168 would not cause GBS cases if a large group of people were infected with it, and there remains a doubt about whether it is really an absolute enteritis-only control. Although GB11 and NCTC 11168 are genetically related, we observed that the LOS biosynthesis genes strongly diverged between these two strains. Previous work (9) showed that the C. jejuni LOS biosynthesis loci of 11 strains could be grouped into three classes (A, B, and C) based on the gene contents in the region from open reading frames (ORFs) Cj1133 (encoding heptosyltransferase I) to Cj1149 (encoding heptosyltransferase II). The sequencing of this region in C. jejuni GB11 showed that it has a class A LOS biosynthesis locus while
Guillain-Barre´ syndrome (GBS) is the most common cause of acute neuromuscular paralysis in countries where poliomyelitis has been eradicated. Miller Fisher syndrome (MFS) is a rare variant of GBS that involves mostly ocular symptoms. Both GBS and MFS are postinfectious neuropathies, and Campylobacter jejuni gastroenteritis is considered to be the most frequent antecedent infection associated with their development (10). The core oligosaccharides of low-molecularweight lipooligosaccharides (LOS) of many C. jejuni strains have been shown to exhibit molecular mimicry of the carbohydrate moieties of gangliosides (2–4). This molecular mimicry between C. jejuni LOS outer-core structures and gangliosides has been suggested to act as a trigger for autoimmune mechanisms in the development of GBS (21). Penner et al. (16) developed a serotyping scheme for C. jejuni based on soluble heat-stable (HS) antigens. There are a few reports of overrepresentation of specific Penner serotypes (HS:19 in Japan and HS:41 in South Africa) among GBSassociated C. jejuni isolates (11, 12). However, the use of various serotyping and molecular typing methods (amplified fragment length polymorphism, multiple locus sequence typing [MLST], pulsed-field gel electrophoresis, and random amplified polymorphic DNA analysis) failed to show any clustering among GBS and MFS isolates from The Netherlands and Belgium (6–8). Although no clustering was observed when the * Corresponding author. Mailing address: Institute for Biological Sciences, National Research Council Canada, 100 Sussex Dr., Ottawa, Ontario, Canada K1A 0R6. Phone: (613) 991-9956. Fax: (613) 9411327. E-mail:
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FIG. 1. DNA sequence comparison of the regions from Cj1130c to Cj1156 in C. jejuni GB11 (GenBank accession number AY422197), NCTC 11168 (GenBank accession number AL139077), and ATCC 43446 (GenBank accession number AF167344) strains. The numbers between the loci indicate the number of base differences in each region. The arrows at the bottom indicate the positions of some of the genes in C. jejuni NCTC 11168 (identified by their Cj numbers). Black arrows represent genes specific to C. jejuni NCTC 11168 (Cj1137c and Cj1138 encode putative glycosyltransferases while Cj1144c and Cj1145c encode proteins with unknown functions). The white arrows represent a gene (orf11a, encoding a putative acetyltransferase) that is found only in C. jejuni GB11 and ATCC 43446. Black- and white-striped arrows represent genes present in all three strains.
NCTC 11168 has a class C LOS biosynthesis locus. We extended the sequencing to the regions upstream and downstream of the LOS biosynthesis loci (from Cj1130c to Cj1156) in both GB11 (GenBank accession number AY422197) and ATCC 43446 (GenBank accession number AF167344). The region from Cj1132c to Cj1149c is nearly identical in GB11 and ATCC 43446, with a difference of only 6 bp over a region of 14,260 bp. The corresponding region in NCTC 11168 has diverged considerably, with four ORFs that are absent in GB11 and ATCC 43446, while the latter pair have one ORF unique to them (Fig. 1). C. jejuni ATCC 43446 is the HS:19 Penner type strain and belongs to the ST-22 clonal complex, while the sequence of its flaA SVRs is only 79% identical to the corresponding sequences in GB11 and NCTC 11168 (which are identical). Other molecular typing methods (amplified fragment length polymorphism, pulsed-field gel electrophoresis, and random amplified polymorphic DNA analysis) also showed that C. jejuni GB11 is closer to NCTC 11168 than to ATCC 43446 (6, 7; also data not shown). Consequently, the presence of the same LOS locus in both ATCC 43446 and GB11 is probably the result of horizontal gene transfer. While the GB11 region from Cj1132c to Cj1149c is nearly identical to that in ATCC 43446, the 5⬘ (Cj1130c to Cj1131c) and 3⬘ (Cj1153c to Cj1156) regions are almost identical to those in NCTC 11168, with no differences (over 2,370 bp) and eight differences (over 4,433 bp), respectively. The near identity between GB11 and NCTC 11168 in these regions and the overall genetic relatedness of these two strains indicate that an A class LOS biosynthesis locus was transferred from an HS:19 strain to GB11 rather than in the other direction. The recombination event involved at least 14.26 kb, but the exact sites of insertion are unclear. There are transition regions that are unique to each strain between the middle region of near identity between GB11 and ATCC 43446 and the 5⬘ and 3⬘ regions of near identity between GB11 and NCTC 11168. The high divergence among the three strains in the transition regions may be the result of multiple independent crossover events.
This could easily explain the 0.3-kb transition region found at the 5⬘ end of Cj1131c (encoding a UDP-glucose 4-epimerase). The other transition region spans four genes (from Cj1149c to Cj1152c) encoding enzymes that are all involved in heptose biosynthesis (www.sanger.ac.uk/Projects/C_jejuni/). This transition region is fairly large (3 kb), and GB11 may also have acquired part of it from another strain in a separate recombination event. MLST studies have shown that C. jejuni strains are genetically diverse, with a weakly clonal population structure, and that horizontal genetic exchange is common (6, 18). C. jejuni is naturally competent (20), and de Boer et al. (5) have shown that genetic exchanges between C. jejuni strains can occur during colonization of chickens (a natural reservoir for Campylobacter). The strains that belong to the HS:19 Penner type seem to be an exception as they were suggested to comprise a clonal population based on various molecular typing methods (13, 14). de Boer et al. (P. de Boer, B. Duim, J. P. M. van Putten, and J. A. Wagenaar, abstract from the 12th International Workshop on Campylobacter, Helicobacter, and Related Organisms, Int. J. Med. Microbiol. 291[S31]:74, 2001) suggested that clonal complexes of C. jejuni are genetically preserved by lack of natural transformation. The horizontal transfer of an LOS locus from an HS:19 strain to an HS:2 strain (GB11) is thus consistent with the observation that genetic exchange is common in C. jejuni although an HS:19 strain is more likely to act as a donor rather than a recipient. The near sequence identity between the ATCC 43446 and GB11 LOS loci does not demonstrate that ATCC 43446 was necessarily the HS:19 donor but is a reflection of the high level of homogeneity among all HS:19 strains. The LOS biosynthesis loci have been sequenced in four HS:19 strains (GenBank accession numbers AY297047, AF130984, AF167345, and AF167344), and they share above 99% DNA sequence identity. We used capillary electrophoresis coupled with electrospray mass spectrometry to obtain data on the LOS structure of
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FIG. 2. LOS outer-core structures expressed by the C. jejuni strains studied in this work (2–4, 17). (A) C. jejuni ATCC 43432 (HS:4 type strain), ATCC 43446 (HS:19 type strain), and GB11. In this structure the terminal Neu5Ac is variable, giving a GD1a mimic when present and a GM1a mimic when absent. (B) C. jejuni NCTC 11168 (the terminal Gal is variable). (C) C. jejuni ATCC 43430 (HS:2 type strain). PEA, phosphorylethanolamine.
GB11 (data not shown). The O-deacylated LOS of GB11 gave an array of masses due to N-linked fatty acid and phosphate variability as observed previously with other C. jejuni samples (19). The mass profile of the GB11 LOS was the same as the ones observed for ATCC 43432 (HS:4 type strain) and ATCC 43446 (HS:19 type strain), two strains which express a mixture of GM1a and GD1a mimics (Fig. 2) and have class A LOS loci. The serum of the GB11-infected patient was previously shown to react with the LOS of C. jejuni GB11 (1). It showed no reaction with the LOS from NCTC 11168 and a strong reaction with the LOS from ATCC 43446 (Fig. 3), which is consistent with GB11’s having an LOS locus similar to that of ATCC 43446 (class A locus) rather than to that of NCTC 11168 (class C locus). The GB11-infected serum also reacted with the LOS of ATCC 43432 (HS:4), which is another strain that has a class A locus and an LOS structure identical to those in ATCC 43446 and GB11 (2). The GB11-infected serum had no reaction with the ATCC 43430 (HS:2) strain, which has a C class locus and an LOS structure related to the one observed in NCTC 11168, although it is more truncated (3). Some studies have shown that HS:19 strains are more commonly associated with GBS than other serotypes (11), and it is tempting to speculate that GB11 acquired the ability to cause GBS following the acquisition of the class A locus present in HS:19 strains. Nachamkin et al. (15) have observed a strong association of
FIG. 3. Specific immunodetection of the C. jejuni GB11 and ATCC 43446 (HS:19 type strain) LOS by using the serum from the GB11infected patient. C. jejuni ATCC 43432 (HS:4 type strain) provides another example of a positive reaction, and ATCC 43430 (HS:2 type strain) provides an example of a negative reaction. Proteinase K-treated preparations of C. jejuni GB11 (lanes 1), ATCC 43430 (lanes 2), ATCC 43432 (lanes 3), NCTC 11168 (lanes 4), and ATCC 43446 (lanes 5) were electrophoresed on deoxycholate–16.5% polyacrylamide gel. (A) The gel was stained using silver stain. (B) The LOS was transferred onto a polyvinylidene difluoride membrane. The membrane was then probed using serum, diluted 1/5,000, from the GB11-infected patient. The secondary antibody was alkaline phosphatase-conjugated rabbit anti-human immunoglobulin G, and the detection was with nitroblue tetrazolium-BCIP (5-bromo-4chloro-3-indolylphosphate).
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three class A genes (cstII, cgtA, and cgtB) with C. jejuni strains isolated from GBS patients. However, more GBS and MFS C. jejuni strains will have to be analyzed to demonstrate a statistically significant association of the class A LOS biosynthesis locus with postinfectious neuropathies. In any case, the reactivity of the serum from the GB11-infected patient against the GB11 LOS and gangliosides (1) certainly substantiates the need for further studies of this LOS locus in other GBS- and MFS-associated strains. Nucleotide sequence accession number. The sequence of the C. jejuni GB11 LOS biosynthesis locus and surrounding regions has been deposited in GenBank under accession number AY422197. This work was supported by the NRC Genome Sciences and Health Related Research Initiative and a research grant from the Human Frontier Science Program. We thank Anna-Maria Cunningham for performing the DNA sequencing, Denis Brochu for editing the sequence, and Frank St. Michael and Rachel Verhulp for technical help.
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