The crucial role of Campylobacter jejuni genes in anti-ganglioside antibody induction in Guillain-Barre syndrome

Abstract

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

Figure 1

Genetic organization of the 5 different classes of the C. jejuni LOS biosynthesis locus. The distance between the scale marks is 1 kb. The direction of the arrows indicates the direction of transcription. Corresponding homologous genes have the same number with a letter for the LOS locus class added. For orf1c and orf13c the corresponding Cj gene numbers of the “genome” strain NCTC 11168 are given. The 5 LOS classes are based on DNA sequences of the following strains (GenBank accession number): class A: OH4384 (AF130984), OH4382 (AF167345), HS:4 (AF215659), HS:10 (AF400048), HS:19 (AF167344), HS:41 (AY044868); class B: HS:23 (AF401529), HS:36 (AF401528); class C: NCTC 11168 (AL139077), HS:1 (AY044156), HS:2 (AF400047); class D: LIO87 (AF400669); class E: 81116 (AF343914 and AJ131360). The proposed functions for the orfs are described in Supplemental Table 1.

Figure 2

Schematic representation of the mutants used in this study. Orf6 to orf13 of the class A LOS locus are displayed. The positions of the 0.7-kb Cm^r cassette are indicated with gray arrows. Both orf10 and orf11 are inactivated in the orf10/orf11 mutant.

Figure 3

Molecular mimicry between gangliosides and LOS outer cores of WT and mutant C. jejuni strains. LOS outer core structures of GB2 and GB11 WT and mutant strains were determined by mass spectrometry analysis. The LOS outer core structure of the “genome” strain NCTC 11168 was described by St. Michael et al. (35). Note that all strains express a mixture of different LOS structures. The cst-II and orf10/orf11 mutants of GB2 and GB11 expressed not ganglioside mimics but a mixture of 3 nonsialylated structures. No structural differences were observed between the WTs and the orf11 mutants.

Figure 4

SDS-PAGE analysis of LOS from GB11 WT and mutants. The genome strain NCTC 11168 was included as a control. Lane 1, WT GB11; lane 2, GB11 cst-II mutant; lane 3, GB11 orf10/orf11 mutant; lane 4, GB11 orf11 mutant; lane 5, NCTC 11168. (A) Silver staining of the LOS revealed faster-migrating LOS cores for the GB11 cst-II and orf10/orf11 mutants compared with the WT, indicating that these mutants have a truncated LOS. The orf11 mutant LOS showed migration patterns identical to those of WT LOS. (B) A Western blot incubated with GB11 patient serum showed a reduced reactivity for the cst-II and orf10/orf11 mutants but unchanged reactivity for the orf11 mutant when compared with the WTs. (C) For the cst-II and orf10/orf11 mutants, the reactivity with cholera toxin, a ligand for GM1-oligosaccharide structures, was almost completely lost. Reactivity with the orf11 mutant remained unchanged.

Figure 5

Anti-glycolipid antibodies in serum of mice before and after immunization with WT and mutant GB11 C. jejuni strains. (A and D) When sialic acid was present on the LOS core (WT and orf11 mutant), anti-GD1a antibodies were induced in mice. (B and C) Anti-GD1a antibody responses were not detected in mice immunized with nonsialylated LOS (cst-II and orf10/orf11 mutants). (F and G) Nonsialylated LOS (cst-II and orf10/orf11 mutants) induced high asialo-GM1 antibody responses. (E and H) Asialo-GM1 antibody responses were absent or low after immunization with the orf11 mutant and WT.