Structure of Campylobacter jejuni lipopolysaccharides determines antiganglioside specificity and clinical features of Guillain-Barré and Miller Fisher patients

Ganglioside mimicry in the lipopolysaccharide (LPS) fraction of Campylobacter jejuni isolated from Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) patients was compared with isolates from patients with an uncomplicated enteritis. The antibody response to C. jejuni LPS and gangliosides in neuropathy patients and controls was compared as well. LPS from GBS and MFS-associated isolates more frequently contained ganglioside-like epitopes compared to control isolates. Almost all neuropathy patients showed a strong antibody response against LPS and multiple gangliosides in contrast to enteritis patients. Isolates from GBS patients more frequently had a GM1-like epitope than isolates from MFS patients. GQ1b-like epitopes were present in all MFS-associated isolates and was associated with anti-GQ1b antibody reactivity and the presence of oculomotor symptoms. These results demonstrate that the expression of ganglioside mimics is a risk factor for the development of post-Campylobacter neuropathy. This study provides additional evidence for the hypothesis that the LPS fraction determines the antiganglioside specificity and clinical features in post-Campylobacter neuropathy patients.     

Molecular characterization of Campylobacter jejuni from patients with Guillain-Barré and Miller Fisher syndromes

Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The risk of developing GBS or MFS may be higher after infection with specific C. jejuni types. To investigate the putative clonality, 18 GBS- or MFS-related C. jejuni strains from The Netherlands and Belgium and 17 control strains were analyzed by serotyping (Penner and Lior), restriction fragment length polymorphism analysis of PCR products of the flaA gene, amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. Serotyping revealed 10 different O serotypes and 7 different Lior serotypes, thereby indicating a lack of serotype clustering. Two new O serotypes, O:35 and O:13/65, not previously associated with GBS or MFS were found. Serotype O:19 was encountered in 2 of 18 strains, and none was of serotype O:41. The results of all genotypic methods also demonstrated substantial heterogeneity. No clustering of GBS- or MFS-related strains occurred and no molecular marker capable of separating pathogenic GBS or MFS from non-GBS- or non-MFS-related enteritis strains could be identified in this study. Sialic-acid-containing lipopolysaccharides (LPS) are thought to be involved in the triggering of GBS or MFS through molecular mimicry with gangliosides in human peripheral nerves. Therefore, further characterization of GBS- or MFS-related C. jejuni should target the genes involved in the synthesis of LPS and the incorporation of sialic acid.     

Campylobacter jejuni enteritis and Guillain-Barré syndrome]

Some patients developed Guillain-Barré syndrome after the administration of bovine brain ganglioside. Patients with Guillain-Barré syndrome subsequent to Campylobacter jejuni enteritis frequently have IgG antibody to GM1 ganglioside. Miller Fisher syndrome, a variant of Guillain-Barré syndrome, is associated with IgG antibody to GQ1b ganglioside. My colleagues and I showed the existence of molecular mimicry between GM1 and lipopolysaccharide of C. jejuni isolated from a patient with Guillain-Barré syndrome, and that between GQ1b and C. jejuni lipopolysaccharides from patients with Miller Fisher syndrome. The glycotope mimicry between infectious agents and gangliosides may function in the production of antiganglioside antibodies and the development of Guillain-Barré syndrome and Miller Fisher syndrome.     

Epidemiology of Campylobacter jejuni isolated from patients with Guillain-Barré and Fisher syndromes in Japan

Campylobacter jejuni isolation is the standard for the diagnosis of this type of bacterial infection, but there have been no epidemiological studies of a large number of C. jejuni isolates from patients with Guillain-Barre syndrome (GBS) and Fisher syndrome (FS). For 13 years, stool specimens from GBS/FS patients have been sent from 378 hospitals throughout Japan to the Tokyo Metropolitan Institute of Public Health. A total of 113 strains (11%) were isolated from the stool specimens from 1,049 patients. The isolation rate did not differ by region. The rates were 22% for 449 patients with a history of diarrhea and 2% for the others. An additional 18 isolates were provided by various hospitals. There was no noticeable seasonal distribution in the onset of C. jejuni isolated from patients with GBS/FS. The male/female ratios were 1.7:1 for GBS and 2.2:1 for FS. The patient age range showed a peak in 10- to 30-year-old subjects who had GBS and in 10- to 20-year-old subjects who had FS. The predominance of young adults and male patients who had C. jejuni-associated GBS/FS may be related to the preponderance of young adults and male patients who had C. jejuni enteritis. The median interval from diarrhea onset to neurologic symptom onset was 10 days for GBS/FS. Penner's C. jejuni serotype HS:19 was more frequently present in GBS (67%) than in enteritis (6%) patients. HS:2 was more frequent in FS (41%) than in enteritis (14%) patients. These findings suggest that certain C. jejuni strains specifically trigger GBS and that others specifically trigger FS.     

Rapid method for sensitive screening of oligosaccharide epitopes in the lipooligosaccharide from Campylobacter jejuni strains isolated from Guillain-Barré syndrome and Miller Fisher syndrome patients

Campylobacter jejuni lipooligosaccharide (LOS) can trigger Guillain-Barré syndrome (GBS) due to its similarity to human gangliosides. Rapid and accurate structural elucidation of the LOS glycan of a strain isolated from a GBS patient could help physicians determine the spectrum of anti-ganglioside antibodies likely to be found and therefore provide valuable assistance in establishing an appropriate course of treatment. The ability of implemented mass spectrometry-based approaches in a clinical setting has been limited by the laborious and time-consuming nature of the protocols, typically 3 to 4 days, used to prepare LOS. In order to improve the analytical throughput, microwave-assisted enzymatic digestion was investigated. In this study, the bacterial cells were suspended in 50 μl of 20 mM ammonium acetate buffer containing DNase and RNase and treated by direct microwave irradiation for 3 min. Then, proteinase K was added and the samples were again microwaved. The intact LOS samples were analyzed using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry. The reliability of the rapid, high-throughput technique was demonstrated through analysis of LOS glycans from 73 C. jejuni strains. The structure was elucidated using material from a single colony. The total time for sample preparation and MS analysis is less than 60 min.     

Prevalence and characteristics of Guillain-Barré syndromes associated with Campylobacter jejuni and cytomegalovirus in greater Paris

AIM OF THE STUDY: We aimed to study prevalence and features of Campylobacter jejuni and cytomegalovirus (CMV)-associated Guillain-Barré syndromes (GBS) in a French care unit. PATIENTS AND METHODS: We studied 264 patients with GBS admitted at Raymond Poincaré hospital (Garches) between 1996 and 2001. Clinical data were obtained prospectively. Sera were collected at patients entry and tested retrospectively for anti-C. jejuni, anti-CMV and antigangliosides GM1 et GM2 antibodies. RESULTS: GBS associated with a serological evidence for a recent C. jejuni infection were the more frequent (58/264, 22%); they affected predominantly men of mature years (mean age: 51.3 years; sex-ratio M/F: 1.76), mostly after a gastrointestinal illness (52%); they were often pure motor forms (57%), were severe (mechanical ventilation: 40%) and associated to an anti-GM1 IgG and/or IgM response (44%). GBS cases involving a primary CMV infection were less frequent (40/264, 15%), but were severe too (mechanical ventilation: 37.5%); they occurred preferentially in young women (mean age: 35.9 years; sex-ratio MF: 0.82), often after respiratory tract symptoms (28%) or an influenza-like syndrome (15%) and were frequently associated with sensory loss (73%) and with an anti-GM2 IgM response (47%). CONCLUSION: C. jejuni and CMV proved to be major triggering agents of GBS in France. They are associated with distinct presentations, which are both severe.     

Siglec-7 specifically recognizes Campylobacter jejuni strains associated with oculomotor weakness in Guillain–Barré syndrome and Miller Fisher syndrome

Due to molecular mimicry, Campylobacter jejuni lipo-oligosaccharides can induce a cross-reactive antibody response to nerve gangliosides, which leads to Guillain–Barré syndrome (GBS). Cross-reactive antibodies to ganglioside GQ1b are strongly associated with oculomotor weakness in GBS and its variant, Miller Fisher syndrome (MFS). Antigen recognition is a crucial first step in the induction of a cross-reactive antibody response, and it has been shown that GQ1b-like epitopes expressed on the surface of C. jejuni are recognized by sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7). We aimed to determine the epitope specificity of C. jejuni binding to Siglec-7, and correlate the outcome to disease symptoms in GBS and MFS patients. Using a well-defined GBS/MFS-associated C. jejuni strain collection, which included three sialic acid knockout strains, we found that Siglec-7 exclusively binds to C. jejuni strains that express terminal disialylated ganglioside mimics. When serological and diagnostic patient records were correlated with the Siglec-7-binding properties, we observed an association between Siglec-7 binding and the presence of anti-GQ1b antibodies in patient serum. In addition, Siglec-7 binding was associated with oculomotor weakness in GBS and MFS patients. Lipo-oligosaccharide-specific binding of C. jejuni to Siglec-7 may be an initiating event in immune recognition and presentation, and lead to anti-GQ1b antibody production and the development of ocular weakness in GBS or MFS.     

Improved serological diagnosis stresses the major role of Campylobacter jejuni in triggering Guillain-Barré syndrome

Guillain-Barré syndrome (GBS) is a postinfectious autoimmune polyradiculoneuropathy. The most frequent antecedent pathogen is Campylobacter jejuni, followed by cytomegalovirus. However, more than 40% of GBS cases currently cannot be attributed to triggering events. This might be due to the shortcomings of the serological assays used for diagnosing infections, in particular for C. jejuni. In our study investigating 36 patients with acute GBS, standard serological methods identified the triggering viral or bacterial etiology in only 25% of cases. However, using a highly specific enzyme-linked immunosorbent assay based on two recombinant outer antigens encoded by C. jejuni genes Cj0017 (P39) and Cj0113 (P18), we found serological evidence of a preceding C. jejuni infection in 80.6% of the patients but in only 3.5% of the controls. We conclude that the role of C. jejuni in triggering GBS has been greatly underestimated.     

Genetic characterization of Campylobacter jejuni O:41 isolates in relation with Guillain-Barré syndrome

Campylobacter jejuni O:41 strains are found in association with Guillain-Barré syndrome in South Africa. Strains of this serotype collected over 17 years were characterized by amplified fragment length polymorphism and flagellin typing to determine their clonal nature. Despite minor variation in GM1 expression, all of the strains were genetically indistinguishable, indicating that they are representative of a genetically stable clone.     

Lymphocyte transformation test detects a response to Campylobacter jejuni antigens in patients with Guillain-Barré syndrome

Guillain-Barré syndrome (GBS) is an immune-mediated polyneuropathy. Campylobacter jejuni-associated gastrointestinal infection is identified as a major precipitating agent of GBS; however, a standard test to diagnose this infection in patients with GBS is lacking. The aim of the present study was to evaluate an outer membrane protein (OMP)-based lymphocyte transformation test (LTT) for the diagnosis of C. jejuni infection in GBS. Forty patients with GBS, age and gender matched 52 healthy controls (HC) and 46 disease controls (DC) were analyzed for C. jejuni infection by culture, polymerase chain reaction (PCR) and LTT. Lymphocytes at concentration of 1 × 10⁶/well isolated from GBS patients and controls were stimulated with 20 μg/ml of C. jejuni OMP, and ³H-thymidine was incorporated to measure cell proliferation. LTT detected significantly higher C. jejuni infection compared to culture (77.5 vs. 2.5%; P < 0.05) and PCR (77.5 vs. 22.5%; P < 0.05). The cutoff value of lymphocyte proliferation by receiver operating characteristic (ROC) curve of 2.5 had 77.5% sensitivity and 96.5% specificity. Area under ROC curve was 0.92. The mean SI of the cell proliferation for GBS cases was significantly higher than the controls (GBS vs. HC; P < 0.001, GBS vs. DC; P < 0.001). LTT appears to be a sensitive tool for detecting preceding C. jejuni infection in GBS patients with reasonable sensitivity and specificity. It is possible that the activated lymphocytes might play role in the pathogenesis of neuronal damage in GBS.     

Comparison of Campylobacter jejuni isolates implicated in Guillain-Barré syndrome and strains that cause enteritis by a DNA microarray

We asked whether Campylobacter jejuni isolated from patients with Guillain-Barré syndrome (GBS) differ from isolates isolated from patients with uncomplicated gastrointestinal infection using DNA microarray analysis. We found that specific GBS genes or regions were not identified, and microarray analysis confirmed significant genomic heterogeneity among the isolates.     

Detection of Campylobacter jejuni by Culture and Real-Time PCR in a French Cohort of Patients with Guillain-Barré Syndrome

Bacteriological culture and real-time PCR (RT-PCR) were used to detect Campylobacter jejuni in fecal samples from a French cohort of 237 patients with Guillain-Barré syndrome (GBS). We provide evidence that diverse serotypes and genotypes of C. jejuni are a major trigger of GBS in France.Guillain-Barré syndrome (GBS) is a postinfectious neurological disease, with a median estimated annual incidence of 1.3/100,000 in developed countries (7). Campylobacter jejuni is the most frequent triggering agent, accounting for 15 to 40% of GBS cases in North America, Australia, and Europe (6, 14, 16, 20). The majority of cases of C. jejuni-associated GBS in Japan involve serotype O:19 (18), and the majority of cases in South Africa involve serotype O:41 (12). A much greater diversity of serotypes (4, 17) and genotypes (4) has been reported in Europe. The pathogenesis of C. jejuni-associated GBS has been linked to antiganglioside autoantibodies generated as a result of the molecular mimicry displayed by ganglioside-like oligosaccharides present in the lipopolysaccharide of certain C. jejuni strains (21). More recently, it has also been linked to the presence of particular classes of the lipo-oligosaccharide (LOS) biosynthesis locus, with most GBS strains having class A or B loci (10, 17).Little is known about the role of C. jejuni as a triggering agent of GBS in France. In a study of 263 GBS cases in the greater Paris area, France, between 1996 and 2001, serological evidence of recent C. jejuni infection was shown in 22% of patients (16). However, no study has provided direct bacteriological evidence of the role of C. jejuni in French GBS cases, and there are no data on the C. jejuni strains involved. We thus undertook a prospective study on a French cohort of patients with GBS based on the detection of C. jejuni in fecal samples by culture and real-time PCR (RT-PCR).The medical intensive care unit at the Raymond Poincaré Hospital (Garches, France) is a regional reference center for the management of adult patients with GBS. Since November 1999, all admitted GBS cases have been tested for the presence of C. jejuni in fecal samples (rectal swab and/or stool samples) by culture and RT-PCR, and anti-C. jejuni antibodies have been detected by the complement fixation test (CFT). All the patients included in this study were patients with GBS (2) who were admitted between November 1999 and December 2005 and who had (i) at least one fecal sample (rectal swab or stool sample) examined for the presence of C. jejuni by culture and RT-PCR and (ii) measurement of anti-C. jejuni antibodies. The clinical data were collected on inclusion in the study as described previously (16). Serum samples and rectal swabs were taken from patients on admission. The patients'' first stools during their hospitalization were also sampled and studied for C. jejuni culture and RT-PCR. Our ethics committee waived the requirement for informed consent because both the screening of fecal samples for C. jejuni and the measurement of anti-C. jejuni antibodies are routinely carried out and do not affect decisions concerning treatment.Stool samples were cultured with blood-containing Campylosel (bioMérieux, Marcy l''Etoile, France) (November 1999 to May 2002) or Butzler medium (Bio-Rad Laboratories, Hercules, CA) (after May 2002) with or without prior enrichment in Preston selective broth (Oxoid, Basingstoke, Hampshire, England) at 42°C. Rectal swabs were immersed in Preston enrichment broth, which was incubated at 42°C for 24 h before culture on selective medium. Campylobacter isolates were identified to the species level at the French National Reference Center for Campylobacter and Helicobacter. Strains were serotyped with the Penner O scheme (13). Genotyping involved sequence analysis of the short variable region of the flaA gene (flaA SVR) (4) and the Campylobacter Fla database (http://pubmlst.org/campylobacter/flaA/; hosted at the University of Oxford). LOS biosynthesis loci were classified as previously described (10). Campylobacter DNA was amplified from stool and rectal samples by a custom-designed RT-PCR technique using the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany). Bacterial DNA from stool samples was purified by using the QIAamp stool DNA minikit (Qiagen GmbH, Germany), and DNA from rectal samples was purified by using the QIAamp DNA minikit (Qiagen). Purified DNA (2 μl) was subjected to amplification with the 16S DNA primers C16S-F (5′-CTAGCTTGCTAGAACTTAGA-3′) and C16S-R (5′-GTCCACACCTTCCTCCTC-3′). We used the LightCycler FastStart DNA master hybridization probe kit (Roche Diagnostics) with fluorescent hybridization probes LC-Camp16S-A (5′-ACGTATTTAGTTGCTAACGGTTC-fluorescein-3′) and LC-Camp16S-B (5′-LightCycler Red 640-GAGCACTCTAAATAGACTGCCTTC-P-3′), synthesized by Proligo (Paris, France). The specificity of amplification was checked by melting curve analysis (melting peaks for Campylobacter species as follows: C. jejuni and C. coli, 64°C with a shoulder effect at 57°C; C. fetus, 57°C; and C. lari, 54°C). Results are expressed qualitatively, as positive or negative (estimated detection threshold of 2 × 10² CFU/g of feces). Serum antibodies against C. jejuni were assayed with complement fixation tests (CFTs) (Virion\Serion, Würzburg, Germany), using a cutoff titer of 20 (>95% specificity; Virion\Serion). Antibodies (IgM/IgG) against gangliosides GM1, GM2, GD1a, GD1b, and GQ1b were detected by enzyme immunoassays (GanglioCombi; Bühlmann Laboratories AG, Schönenbuch, Switzerland). Statistical analyses were performed with R. 2.8.1 software (The R Foundation for Statistical Computing, Vienna, Austria). Categorical variables were compared using Fisher''s exact tests, and quantitative variables were compared using Wilcoxon tests. All tests were two tailed, and P values of <0.05 were considered significant.We included 237 patients with GBS in this study (Table ​(Table1).1). All patients were assessed for the presence of C. jejuni in fecal samples by culture and RT-PCR (rectal swabs alone, 133 patients; stool samples alone, 23 patients; both samples, 81 patients) and for the presence of serum anti-C. jejuni antibodies with CFT. Sixteen patients (6.8%) gave positive test results with RT-PCR and/or culture; of these, eight were positive by both methods, six were positive by RT-PCR alone, and two were positive by culture alone. C. jejuni was isolated from nine patients, and Campylobacter coli was isolated from one patient. The CFT was positive in 63 patients (26.6%), which included 15 of the 16 patients with positive RT-PCR and/or culture results. Thus, in total, 64 were shown to be associated with C. jejuni (or C. coli) from the 237 patients studied (27%). These cases were more likely to be male, have prodromal diarrhea, be admitted a few days after GBS onset, and have antiganglioside antibodies and a pure motor form than cases with no evidence of recent C. jejuni infection (n = 173) (Table ​(Table11).

TABLE 1.

Characteristics of the 237 patients studied^a

Flagella as a potential marker for Campylobacter jejuni strains associated with Guillain-Barré syndrome

Campylobacter jejuni recovered from patients with Guillain-Barré syndrome (GBS) in different geographical locations and bearing different heat-labile and heat-stable antigens were found to have identical amino acid sequences in their flagellar flaA short variable region, suggesting that it may be a potentially useful marker for GBS association.     

Campylobacter jejuni from patients with Guillain-Barré syndrome preferentially expresses a GD(1a)-like epitope

GM(1)- and GD(1a)-like ganglioside mimicry in Campylobacter jejuni lipooligosaccharide (LOS) is considered to be involved in the pathogenesis of Campylobacter-induced Guillain-Barré syndrome (GBS). Compared with gastroenteritis-related isolates, GBS-related C. jejuni isolates were strongly associated with the expression of GD(1a)-like mimicry. The presence of a few genes involved in LOS ganglioside mimicry, cst-II, cgtA, and cgtB, was also associated with GBS-related strains. GD(1a)-like epitope expression may be an important virulence phenotype associated with the risk of developing GBS following campylobacter infection.     

PCR-restriction fragment length polymorphism analysis of Campylobacter jejuni genes involved in lipooligosaccharide biosynthesis identifies putative molecular markers for Guillain-Barré syndrome

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.     

Sequence typing confirms that Campylobacter jejuni strains associated with Guillain-Barré and Miller-Fisher syndromes are of diverse genetic lineage, serotype, and flagella type

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.     

Anti-GD1b IgG positive case of overlapping Ficher's and Guillain-Barré syndromes

We describe a patient who developed overlapping sensory ataxic form of Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) following Campylobacter jejuni infection. Two cerebrospinal fluid examinations shown albuminocytologic dissociation associated with Campylobacter jejuni infection after tongue pierced. He had high titers of monospecific anti-GD1b IgG antibody. Because of the rarety of this disorder the diagnostic was difficult. There is a close association of IgG anti-ganglioside GD1b antibodies in sensory ataxic GBS. The findings of the present study show that antibody to GD1b ganglioside is one of the immunological factors in the pathogenesis of sensory ataxic form of GBS, a rare specific immuno-clinical variant form of GBS with prominent sensory ataxia.     

Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome

Guillain-Barré syndrome following Campylobacter jejuni infection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority of Campylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.