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 This strain has been shown to cause an inflammatory diarrhea in two human feeding studies (8; D. Tribble, unpublished data) and to cause disease in experimental models using primates (40) and ferrets (19,67).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 ...

SummaryCampylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a highmolecular-weight (HMW) glycan (apparent M r 26 kDa to . 85 kDa) and increased resolution of a second ladder-like glycan (apparent M r 26±50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsMindependent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an a2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant insertional mutants of C. jejuni 81-176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2-37 and K2-55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2-37 and K2-55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90,977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.

Campylobacter jejuni 81-176 pgl mutants impaired in general protein glycosylation showed reduced ability to adhere to and invade INT407 cells and to colonize intestinal tracts of mice.There is an increasing awareness of the existence of prokaryotic glycoproteins (36), often in complex surface structures such as pili (7,8,28,39), S layers (37), and flagella (6,10,11,12,19,23,44). Among glycosylated flagellins, those of Campylobacter spp. are the best characterized (11,16,42). The nature and extent of flagellin glycosylation have been determined for strain 81-176, one of the best-characterized strains of Campylobacter jejuni (2,3,5,21,29,41,45,46) and one which has been documented to cause diarrheal disease in two volunteer feeding studies 5; D. T. Tribble, unpublished data). Flagellin from 81-176 contains 19 sites of O-linked glycosylation to the monosaccharide pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid) and analogs of pseudaminic acid (42). Additionally, C. jejuni 81-176 has been shown to contain a general protein glycosylation (pgl) system affecting many other soluble and membrane-associated proteins (41). The only reported phenotype of pgl mutants has been the loss of immunogenicity of multiple proteins as detected by Western blot analyses using polyclonal, hyperimmune rabbit antisera, changes that were identical to those seen following chemical deglycosylation of the same protein preparations (42). However, neither the identity of the proteins glycosylated by the pgl system nor the chemical nature of the attached carbohydrate(s) has been reported. This study describes additional phenotypes of 81-176 pglB and pglE mutants. The predicted protein encoded by pglB shows significant similarity to domains of an oligosaccharide transferase of Saccharomyces cerevisiae (48) and an ortholog in Methanobacterium spp. (38). PglE shows highest similarity to a putative aminotransferase involved in lipopolysaccharide synthesis in Bacteroides fragilis (9). The protein also shows homology to proteins involved in glycosylation of pilin in Neisseria spp. (20, 31) and flagellin in Caulobacter crescentus (23) and Aeromonas caviae (13,32).Growth comparisons. Cell morphology, as determined by transmission electron microscopy, was similar for 81-176 and pglB and pglE mutants (results not shown). Bacterial growth curves (Fig. 1) indicated that both mutants had slightly faster doubling times relative to 81-176. However, only the pglE mutant demonstrated a statistically significant increase in growth rate (P Ͻ 0.05) compared to the wild type by paired t-test analysis. Complementation of the pglE mutation in trans with plasmid pCS101, an Escherichia coli-Campylobacter shuttle vector containing an intact copy of pglE and its putative promoter (41), restored the wild-type doubling time.Since numerous soluble and membrane proteins appear to be glycosylated by the pgl system, it was possible that the mutants would display increased sensitivity to growth inhibitors. The sensitivity of wild-type 81-1...