Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-Lglycero-L-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.

A genetic locus from Campylobacter jejuni 81‐176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5α containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site‐specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site‐specific mutation of each of the seven genes in 81‐176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild‐type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81‐176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.Although Campylobacter jejuni is one of the major causes of bacterial diarrhea worldwide (51, 59), the details of its molecular pathogenesis are not well understood. The clinical symptoms of campylobacter infection can range from a mild, watery diarrhea to a dysentery-like illness with fecal blood and leukocytes (2). Although there are reports of numerous cytotoxins, only the cytolethal distending toxin, which arrests eukaryotic cells at the G 2 phase of the cell cycle (64), has been characterized in detail. There are numerous reports that C. jejuni strains can invade intestinal epithelial cells in vitro (20,21,24,29,30,38), although levels of invasion by different strains vary considerably (20,28,38,52). Strain 81-176, originally isolated from a diarrheal outbreak associated with raw-milk consumption (31), is one of the best-characterized strains of C. jejuni. This strain has been shown to cause an inflammatory diarrhea in two human feeding studies (8; D. Tribble, unpublished data) and to cause disease in experimental models using primates (40) and ferrets (19,67). Further, C. jejuni strain 81-176 invades INT407 cells at levels higher than those of most other C. jejuni strains (28, 38).Plasmids have been found in between 19 and 53% of C. jejuni strains (5, 9-11, 41, 53-58), and many of these have been reported to be R plasmids that are transmissible among Campylobacter spp. but not to Escherichia coli (53)(54)(55)(56)(57)(58)60). Despite the importance of plasmids to virulence in numerous other pathogens, it is generally believed that plasmids play no role in Campylobacter pathogenicity. This paradigm is based on the rather low level at which plasmids ...

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The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM 2 and GM 3 gangliosides, with minor amounts of structures mimicking GD 1b and GD 2 . Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM 2 to GM 3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM 3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide (10, 25) and has been shown to be frequently associated with the development of Guillain-Barré syndrome (GBS), a postinfectious polyneuropathy (23). The association of C. jejuni and the development of GBS is thought to result from the molecular mimicry between outer core structures of bacterial lipooligosaccharides (LOS) and human gangliosides (18,19). Thus, for example, the cores of different isolates of C. jejuni have been shown to mimic GM 1 , GM 2 , GD 3 , GD 1a , GT 1a , and GQ 1b (1-3, 18-24, 29, 30, 42, 43). However, similar ganglioside mimicry can be found in strains associated with both uncomplicated enteritis and GBS (18). While considerable research efforts have focused on the role of LOS in the development of GBS, little attention has been placed on the function of these sialylated LOS structures in the pathogenesis of gastrointestinal disease. A locus involved in LOS biosynthesis in C. jejuni MSC57360, the type strain of the HS:1 serogroup, has been described previously (12). It has also been shown that the loss of sialic acid (NeuNAc) from the LOS core results in the increased immunogenicity of the core and an increased sensitivity to normal human serum (12). However, strain MSC57360 is noninvasive in vitro (P. Guerry and C. P. Ewing, unpublished data), and little is known about its virulence potential. In this report, we have characterized the LOS core of C. jejuni 81-176 (serogroups HS:23 and HS:36), which is one of the best-characterized strains of C. jejuni (5-7, 14, 15, 26, 28, 41) and one which has been shown to induce diarrhea in human volunteers in two separate studies (7; D. Tribble et al., unpublished data). These results indicate that the LOS core of C. jejuni 81-176 is composed of structures that mimic several gan...