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...

Campylobacter spp. are among the most common causes of human bacterial diarrhea worldwide. While campylobacter infections are quite common and often severe, relatively little is known about mechanisms of pathogenesis. Campylobacters are generally considered invasive, and invasiveness appears to be associated with disease in the ferret diarrheal disease model (3, 42). In addition, numerous cytotoxins in campylobacters have been described (39), but only cytolethal distending toxin (CDT) has been well characterized (29,30,40). CDT has been found in a variety of other bacteria including Escherichia coli (17, 33), Shigella spp. (26), Haemophilus ducreyi (12), Actinobacillus actinomycetemcomitans (25,35,36), and Helicobacter hepaticus (43). Although CDT has been shown to block eukaryotic cells in the G 2 phase of the cell cycle (6,28,40,43), its role in disease caused by such a diverse group of pathogens remains unclear. However, there is some evidence suggesting a role for CDT in diarrheal disease. An epidemiological study in Bangladesh showed a trend towards increased numbers of CDTpositive E. coli cells in diarrheal cases compared to asymptomatic controls, but the difference did not reach statistical significance (1). When fed to suckling mice, partially purified CDT from Shigella dysenteriae produced watery diarrhea and tissue damage in the descending colon (26).Campylobacter entercolitis is typically associated with a local acute inflammatory response and involves intestinal tissue damage. It is thought that the host inflammatory response may mediate many of the clinical symptoms (20), and inflammatory cytokine responses are recognized components of enteric infections. Interleukin-8 (IL-8) is a proinflammatory cytokine, a potent chemotactic factor for many immune effector cells, and a mediator of localized inflammatory responses. Helicobacter pylori, a primary cause of gastritis in humans, is known to induce IL-8 release from epithelial cells (15,34). Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Shigella spp. have also been shown to elicit IL-8 secretion from intestinal epithelial cells in vitro during invasion (9, 19). We have previously shown that Campylobacter jejuni also induces IL-8 secretion from intestinal epithelial cells by a process which correlated with adherence and/or bacterial invasion (14). In this report we demonstrate that C. jejuni mediates IL-8 secretion from intestinal epithelial cells by multiple mechanisms. One of these mechanisms, as previously described, involves adherence and/or invasion (14), while a second mechanism is mediated directly by CDT. MATERIALS AND METHODSBacterial strains and growth conditions. Campylobacters were routinely grown on Mueller-Hinton (MH) agar (Difco, Detroit, Mich.) under microaerobic conditions or in biphasic MH cultures. MH medium was supplemented with kanamycin to a final concentration of 50 g/ml or chloramphenicol at a final concentration of 20 g/ml in some cases. For counterselection of E. coli DH5␣ (RK212.1) donors (11, 21) in comp...

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM 2 ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative ␤-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.Campylobacter jejuni and Campylobacter coli are among the most prevalent causes of bacterial diarrhea in the world (15, 31). These organisms are antigenically complex, as demonstrated by the fact that there are Ͼ70 serotypes based on heat-stable (HS) antigens (34) and Ͼ100 serotypes based on the heat-labile serotyping scheme (26). Campylobacters contain sialic acid moieties both in lipooligosaccharide (LOS) core structures (3-6, 29) and in posttranslational modifications on flagellin (9).Structural analyses of a limited number of campylobacter strains has revealed LOS-like variability in the outer core (28,29). Moreover, the outer cores of strains from multiple serogroups contain sialic acid moieties in structures which mimic human gangliosides. This molecular mimicry is thought to result in an autoimmune response responsible for the association of some campylobacter serotypes with Guillain-Barré syndrome (GBS) (1,28,29). However, the biological function of sialylated LOS to pathogenesis of gastroenteritis by C. jejuni has not been examined experimentally.Campylobacter spp. are capable of endogenous biosynthesis of sialic acid (3-6, 9, 29). The genome of C. jejuni NCTC 11168 contains multiple genes which encode proteins with similarity to prokaryotic enzymes involved in biosynthesis of sialic acid, N-acetylneuraminic acid (NeuNAc) (33). For example, NCTC 11168 has three copies of genes encoding proteins with sequence similarity to sialic acid synthases (25)...

The circular pVir plasmid of Campylobacter jejuni strain 81-176 was determined to be 37,468 nucleotides in length with a G؉C content of 26%. A total of 83% of the plasmid represented coding information, and all but 2 of the 54 predicted open reading frames were encoded on the same DNA strand. There were seven genes on the plasmid in a continguous region of 8.9 kb that encoded orthologs of type IV secretion proteins found in Helicobacter pylori, including four that have been described previously

Cj0859c, or FspA, is a small, acidic protein ofCampylobacter jejunithat is expressed by a σ28promoter. Analysis of thefspAgene in 41 isolates ofC. jejunirevealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novelC. jejunivirulence factor, and the observed heterogeneity amongfspAalleles suggests alternate virulence potential among different strains.