Isolation of motile and non‐motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells

Abstract

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 proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

“…As for many other bacteria, C. jejuni possesses genes essential for chemotaxis, such as cheA, chew, cheY and MCP receptors. Moreover, chemotactic-mediated motility has been shown to be important for Campylobacter colonization (Takata et al, 1992;Yao et al, 1994). Altogether, 10 genes encoding chemotaxis receptor proteins have been detected in the genome of C. jejuni, designated Tlps for transducer-like proteins.…”

“…Motility of C. jejuni has been shown to be an intestinal colonization factor (Morooka et al, 1985), and O-linked glycosylation of flagellin is crucial for the attachment of the bacterium to intestinal epithelial cells (Yao et al, 1994). In addition, the motility of the pathogen is linked to chemotaxis, which was initially described by Hugdahl et al (1988).…”

Is theCampylobacter jejunisecretory protein Cj0069 a suitable antigen for serodiagnostics?

“…The flagellar motility of the bacterium is an important factor for colonization of the intestine of its avian and mammalian hosts and especially for the invasion of intestinal epithelial cells and for overcoming the mucosal barrier [14,17,18,22,30,31,32]. Furthermore, bacterial secretion systems are used for the export of proteins including some virulence-associated factors.…”

Molecular mechanisms and biological role ofCampylobacter jejuniattachment to host cells

“…A number of studies have suggested a role of flagella as an adhesin [30][31][32][33]. In particular, McSweegan and Walker demonstrated that purified flagella specifically adhered to INT407 cells [34].…”