Preplanned Studies: Emerging Sialylated Class R Lipooligosaccharides in Campylobacter jejuni from Seagulls Has the Potential to Trigger Guillain-Barré Syndrome — Yunnan Province, China, 2018–2023

Campylobacter jejuni lipooligosaccharides (LOS) possess a mimicry antigen structure with gangliosides, explaining the mechanism by which C. jejuni causes Guillain-Barré syndrome (GBS) (1). This study investigated 25 C. jejuni strains isolated from seagulls in southern China, 12 of which possessed class R LOSs. Notably, all 12 strains belonged to specific serotypes (HS2, HS4c, HS19, HS41) previously shown to be strongly associated with GBS (2). Further genetic dissection revealed that these 12 strains could be classified into 2 groups based on LOS sialylation: sialylated (8 strains) and non-sialylated (4 strains). Antigenicity analysis using immunized rabbit serum and serum from GBS patients demonstrated that antigen-antibody reactions occurred only in strains with sialylated LOSs, termed here as emerging C. jejuni. These results indicate that emerging C. jejuni strains, possessing both GBS-associated serotypes and sialylated class R LOSs, exhibit ganglioside mimicry antigen structures and a high potential for triggering GBS.

A total of 25 C. jejuni strains isolated from the feces of seagulls in Kunming City, Yunnan Province from 2018 to 2023, and 4 laboratory-stocked GBS-associated strains coded ICDCCJ07001 (HS:41, LOS class A, GD3-mimic), ICDCCJ07002 (HS:41, LOS class A, ganglioside mimic as GT1a, GM1, GD1a, GD1b, GT1b), HB-CJGB-QYT (HS:2, LOS class A, ganglioside mimic as GT1a, GM1, GD1a, GD1b, GT1b), and HB-CJGB-ZB (HS:19, LOS class A, ganglioside mimic as GD1a, GM1) were included in this study (Table 1). Campylobacter spp. isolation was carried out using the Campylobacter Isolation Kit and the membrane filter method (ZC-CAMPY-002, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China). Suspected colonies were picked and identified by Gram stain and biochemical tests. Polymerase chain reaction (Real-time PCR) tests were used to confirm and identify the species according to a previous study (3). The potential for ganglioside mimicry of these four strains was previously determined in Dr. Gilbert’s lab via mass spectrometry analysis (data not shown). All studied strains were serotyped using a set of 25 commercial antisera for the serotyping of heat-stable (Penner) antigens of C. jejuni by the passive hemagglutination method. LOSs for immunization experiments were purified from strains ICDCCJ07001, ICDCCJ07002, HB-CJGB-QYT, and HB-CJGB-ZB by the hot aqueous-phenol method. New Zealand White rabbits (2.0 to 2.5 kg) were immunized with 400 μg of LOS in complete Freund's adjuvant. Blood was collected from the immunized rabbits until the serum titer reached 1∶100,000. Seven serum samples from GBS patients in a GBS outbreak in Jilin (northern China) were stocked in the laboratory (4). Ganglioside antibodies were previously measured, and antibodies against gangliosides GT1a and GM1 were positive. The antigenicity of the LOSs was analyzed using Western blotting.

A total of 13 LOS classes and 12 capsular genotypes were identified in 25 C. jejuni strains (5) (Table 1). GBS-associated capsular serotypes (HS2, HS4c, HS19, HS41, HS1_44, HS23_36) accounted for 64% (16/25) of strains. Besides, 12 of 25 (48%) strains displayed class R LOS. All 12 class R LOS strains possessed GBS-associated capsular genotypes, including HS2 (3/12, 25%), HS4 (4/12, 33.3%), HS19 (1/12, 8.3%), and HS41 (4/12, 33.3%). The phenotypic serotypes were consistent with the CPS genotypes listed in Table 1. The class R locus contains a large portion of the LOS class A locus; the first 9,700 nt showed 97% sequence identity to LOS class A, including orf3r (encoding a two-domain glucosyltransferase, Cj1135), orf4r (Cj1136), orf5r (CgtA), orf6r (CgtB), orf7r (CstII), orf8r (NeuB), orf9r (NeuC), and orf10r (NeuA). However, orf14r is specific to class R and distinguishes class A from class R (Figure 1A). The orf5a1 of strain ATCC43446 encodes CgtA, whose acceptor is a sialylated galactosyl residue, while the CgtA of strain ATCC43438 utilizes a non-sialylated acceptor. These alternative alleles are also present in class R. Based on gene content and organization of the LOS biosynthesis loci from C. jejuni class R strains, the loci were grouped into two major groups: group R1 strains with sialylated LOS (8 strains) and group R2 with non-sialylated LOS (4 strains). Amino acid sequences of CgtA and CgtB are nearly identical (98% identity) between ATCC 43446 and class R1 strains and 99% identical between ATCC 43438 and class R2 strains (Figure 1B and 1C).

Figure 1. 

LOS class R related to class A. (A) Genetic organization of LOS biosynthesis locus of class A and R strains. (B) Alignment of the cgtA and (C) cgtB educed amino acid sequences.

Note: Four genes (orf 1, orf 2, orf 12, orf 13) were previously recognized as responsible for LOS inner core biosynthesis. Eight genes (orf 3 to orf 10, shown in red) have a high identity to LOS class A. There is a gene (orf 14r, shown in blue), unique to class R. Alignment of the cgtA (orf 5) (1B) and cgtB (orf 6) (1C) deduced amino acid sequences showed that cgtA (orf 5) and cgtB (orf 6) of LOS class R strains use either sialylated (99% identity with class A1 strain ATCC43446) or non-sialylated (99% identity with class A2 strain ATCC43438) acceptors, and class R strains were classified into class R1 and class R2.

Abbreviation: LOS=lipooligosaccharides.

Based on the genetic characteristics and previous investigations, the mimic antigen structures of GBS-associated C. jejuni strains ICDCCJ07002, HB-CJGB-QYT，HB-CJGB-ZB, and ICDCCJ07001 were demonstrated in Figure 2A. LOSs from six strains from two groups of class R (group R1 and group R2) were purified and verified with silver staining for PAGE (Figure 2B). The reactions between different class R LOS antigens from different strains and the serum of rabbits immunized with GBS-associated strains were presented in Figure 2C. Serum immunized with LOS of strain ICDCCJ07001 did not react with any of the purified class R LOSs (Figure 2C). LOSs from three class R1 strains showed a strong reaction with the serum from seven antiganglioside-positive GBS patients. None of the R2 LOSs reacted with the patients’ serum. Sera of healthy controls did not react with any of the class R LOSs tested.

Figure 2. 

Reaction of class R LOS with sera of either immunized rabbit or GBS patient. (A) Structures of various C. jejuni LOS used for immunization of rabbits and corresponding ganglioside mimics. (B) Silver staining of the purified class R LOS (three strains from group R1 LOS and group R2 LOS were selected in this figure, respectively). (C) Western blot analysis of class R LOS with sera from LOS-immunized rabbits. (D) Western blot analysis of class R LOS with GBS patient serum.

Note: M, marker. POS, positive control, purified LOS from different strains to immunize rabbits. a LOS from strain KW_CHFB_024 (group R2); b LOS from strain KW_CHFB_009 (group R1); c LOS from strain KW_CHFB_011 (group R1); d LOS from strain KW_CHFB_014 (group R1); e LOS from strain KW_CHFB_017 (group R2); f LOS from strain KW_CHFB_022 (group R2).

Abbreviation: LOS=lipooligosaccharides.