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Journal of Clinical Microbiology, July 2007, p. 2316-2320, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00203-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

PCR-Restriction Fragment Length Polymorphism Analysis of Campylobacter jejuni Genes Involved in Lipooligosaccharide Biosynthesis Identifies Putative Molecular Markers for Guillain-Barré Syndrome

Peggy C. R. Godschalk,^1* Alex van Belkum,¹ Nicole van den Braak,^1, Diana van Netten,¹ C. Wim Ang,^1, Bart C. Jacobs,^2,3 Michel Gilbert,⁴ and Hubert P. Endtz¹

Departments of Medical Microbiology and Infectious Diseases,¹ Immunology,² Neurology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands,³ Institute for Biological Sciences, National Research Council Canada, Ottawa, Canada⁴

Received 26 January 2007/ Returned for modification 18 March 2007/ Accepted 8 May 2007

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Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy.

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Campylobacter jejuni probably triggers the Guillain-Barré syndrome (GBS) through molecular mimicry between lipo-oligosaccharides (LOS) in the bacterial cell wall and gangliosides in human peripheral nerve tissue (1). Various ganglioside-mimicking structures have been identified in the LOS fraction of the C. jejuni cell wall (9). This variation in LOS structure is the result of differences in the presence of LOS biosynthesis genes and of DNA sequence polymorphism within these genes (4). Based on the differences in gene content observed so far, eight different classes of the LOS biosynthesis gene locus can be identified (6, 10). However, only strains with a class A, B, or C LOS locus express ganglioside mimics (3). Previously, we demonstrated that class A and B LOS biosynthesis gene loci are associated with GBS and its variant, the Miller Fisher syndrome (MFS), and with the expression of ganglioside mimics (5). In search of other and/or more specific markers for GBS/MFS or the expression of ganglioside mimics, we describe a study in which the presence and heterogeneity of individual genes within the class A, B, and C LOS loci were studied by a comparative PCR-restriction fragment length polymorphism (RFLP) analysis of neuropathy-associated and control C. jejuni strains.

The C. jejuni strains used in this study have been described before and represent a genetically heterogeneous population (see Table 2) (5, 11). The presence of GM1-like, GQ1b-like, or "any" ganglioside mimics in the LOS of the strains has also been determined previously by mass spectrometry analysis or immunological methodologies (2, 3, 6). GD3-like or GD1c-like LOS structures were considered to be GQ1b-like mimics (6). Only strains with a class A, B, or C LOS locus express ganglioside mimics. Therefore, specific PCR tests were developed for the individual genes within the class A, B, and C LOS loci (Table 1). When necessary, primer sequences were selected for both class C and class A/B genes to cover intrinsic sequence variabilities as effectively as possible. PCR assays were performed using a Biomed thermal cycler (model 60; Theres, Germany) with a program consisting of 40 cycles of the following cycling protocol: 1 min at 94°C, 1 min at 55°C, 1 min at 72°C. For some amplifications, timing needed to be adapted. For RFLP analysis, PCR products were subjected to overnight incubation at 37°C with the enzymes AluI, DdeI, HindIII, and DraI (Boehringer-Mannheim) in separate reactions. Length determination of the PCR and the RFLP products was performed by agarose gel electrophoresis (1 to 3%, depending on the fragment size). Single band differences led to the introduction of a novel type. The differential presence of the genes was further confirmed by hybridization studies. PCR fragments were labeled with an ECL chemiluminescence kit (Amersham Pharmacia Biotech, Freiburg, Germany) according to the instructions of the manufacturer and hybridized to spot blots containing 200 ng of DNA from the various strains. In short, after 2 h of prehybridization, 500 ng of each PCR product was labeled and hybridized overnight at 42°C. After they were washed, blots were incubated for 1 min in 20 ml of detection reagent. Films were developed after 1-, 5-, and 30-min exposures. Statistical analysis was performed with Instat (version 2.05a; GraphPad Software, San Diego, CA). A P value of <0.05 was considered significant.

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TABLE 2. Characteristics of C. jejuni strains and results of the PCR-RFLP and hybridization analyses for the LOS biosynthesis locus

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TABLE 1. Survey of Campylobacter jejuni NCTC 11168 and HS:19 LOS biosynthesis genes, including primers for amplification of the respective genes

The results of the PCR-RFLP and confirmatory hybridization analyses are summarized in Table 2. In 17% of all positive hybridization signals (the percentage varied per gene), we observed a negative corresponding PCR. In these cases, we considered the gene to be present because sequence heterogeneity at the primer site may result in a negative PCR. For strains with a class A, B, or C LOS locus, the gene content, as determined by PCR and hybridization analyses, was largely in accordance with the expected gene content based on the type of LOS locus. However, there were some discrepancies. orf6 could not be detected in 8 out of 34 (24%) strains with a class A, B, or C LOS locus, although its presence was expected based on the type of LOS locus. A possible explanation may be a failure to detect orf6 due to extensive sequence heterogeneity within orf6, as reflected by the large number of different RFLP types (Table 2). On the other hand, it is also possible that orf6 is really absent in these strains. In five strains with a LOS class other than A, B, or C, one or more genes considered to be unique for class A, B, or C strains gave a positive PCR and hybridization signal. Further analysis is needed to determine whether these positive signals were caused by the actual presence of the target genes in the LOS locus or by the presence of the gene (or a homologue) elsewhere in the genome. Indications for both forms of LOS cluster heterogeneity were documented previously by Parker et al. (10).

Table 3 shows the putative association of the various LOS biosynthesis genes with neuropathy. orf11, encoding a sialate-O-acetyltransferase, was detected significantly more frequently in neuropathy-associated strains and particularly in strains associated with ophthalmoplegia (GBS and MFS) than in controls (Table 3). orf10, located next to orf11 and encoding a CMP-sialic acid synthetase, also occurred more frequently in strains associated with ophthalmoplegia than in controls, but the difference was not statistically significant in the total group of neuropathy-associated strains (Table 3). Because both orf10 and orf11 are unique for classes A and B LOS loci, these findings are in accordance with our previous observations that the class A and B LOS loci are associated with neuropathy. We also demonstrated previously that orf10, but not orf11, is necessary for the biosynthesis of ganglioside mimics and the induction of antiganglioside antibodies in mice (5). Very recently, it was demonstrated that the product of orf11 has sialate-O-acetyltransferase activity and catalyzes the transfer of O-acetyl groups onto oligosaccharide-bound sialic acid (7). However, whether or not this sialate-O-acetyltransferase plays a functional role in the pathogenesis of GBS remains to be determined.

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TABLE 3. Association of gene presence with disease potency as determined for GBS/MFS-associated strains versus controlsa

We also investigated the correlation between the presence of individual LOS biosynthesis genes and the expression of ganglioside-like structures. Three genes were associated with the occurrence of a GQ1b-like epitope in the bacterial LOS, orf7ab (cst-II), orf10, and orf11 (Table 4). Thus, the previously described association between the cst-II gene and the expression of a GQ1b-like epitope was confirmed in the present study (8, 11). Furthermore, we found that all genes that are unique for classes A and B or class A, B, and C LOS loci were significantly associated with the expression of a ganglioside mimic (Table 4), which is concordant with the observation that only the class A, B, and C LOS loci contain the genes that are necessary for the biosynthesis of ganglioside mimics.

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TABLE 4. Association of gene presence with the expression of ganglioside mimics on the LOSa

Variation in gene content is only one of the five known mechanisms that allow C. jejuni to vary the structure of its LOS (4). Other mechanisms, which include phase variation due to homopolymeric tract polymorphism and single or multiple mutations, may be detected by RFLP analysis of the LOS biosynthesis genes. This approach enables the screening of large groups of strains for polymorphism in multiple genes. The number of RFLP types that were found in the current study varied strongly per gene (Table 2). Interestingly, most genes in the class C strains were very homogeneous, with only one or two different RFLP types. This is in contrast with the large number of RFLP types that were observed for many genes in the class A and B strains. It is possible that the class C locus has evolved more recently than the less homogeneous class A and B loci (3). Considerable heterogeneity with more than six RFLP types per gene was detected in orf1 (waaC), orf2, orf6 (cgtB), orf7ab (cst-II), orf9 (neuC), orf10 (neuA), orf12, and orf13 (waaF). These genes are involved in different steps of LOS biosynthesis, including the biosynthesis and transfer of sialic acid, a crucial component of gangliosides (Table 1). Such variation on the genetic level may imply functional differences in the products of these genes, leading to qualitative or quantitative variation in expression of ganglioside mimics. It has been demonstrated previously that specific polymorphism in the cst-II gene determines whether the LOS contains mono-NeuAc (the "GM1-like") or di-NeuAc (the "GQ1b-like") ganglioside (4). However, in this study, we did not detect an association between the cst-II RFLP type and the mono- or disialylated LOS (results not shown). This may be due to the limited sensitivity of RFLP analysis compared with that of DNA sequence analysis. For the other genes, we did not find either a specific RFLP type that was associated with neuropathy or the expression of ganglioside mimics (results not shown). It may, therefore, be necessary to perform elaborate DNA sequence analysis to determine the putative role of point mutations in the expression of ganglioside mimics and pathogenesis of post-Campylobacter infection neuropathy.

In conclusion, PCR-RFLP analysis demonstrated considerable variation in gene content and overall sequence heterogeneity in the C. jejuni LOS biosynthesis locus. We confirmed and extended previous observations that specific LOS biosynthesis genes are associated with neuropathy and the biosynthesis of ganglioside mimics. RFLP analysis did not demonstrate sequence heterogeneity within genes that was associated with neuropathy or the expression of ganglioside mimics. This technique may not be suitable to detect such polymorphism due to its limited sensitivity.

 
This study was supported by grants from The Netherlands Organization for Scientific Research (920-03-225) to P.C.R.G. and from the Human Frontier Science Program (RGP 38/2003).

 
* Corresponding author. Mailing address: Department of Medical Microbiology and Infectious Diseases, Erasmus MC, ‘s-Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands. Phone: 31-10-4633510. Fax: 31-10-4633875. E-mail: p.godschalk{at}erasmusmc.nl

Published ahead of print on 16 May 2007.

Present address: Avans University, Breda, The Netherlands.

Present address: Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands.

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Journal of Clinical Microbiology, July 2007, p. 2316-2320, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00203-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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