ISOLATION OF MOTILE AND NONMOTILE INSERTIONAL MUTANTS OF CAMPYLOBACTER-JEJUNI - THE ROLE OF MOTILITY IN ADHERENCE AND INVASION OF EUKARYOTIC CELLS

A method of insertional mutagenesis for naturally transformable organi sms has been adapted from Haemophilus influenzae and applied to the st udy 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 noninva sive mutants were identified which showed 18-200-fold reductions in th e level of invasion compared with the parent. Three of these eight sho w defects in motility, and five are fully motile. The three mutants wi th motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an ins ertion of the kanamycin-resistance cassette into the flaA flagellin ge ne 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 flage llar 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 aff ecting the same gene, termed pflA (paralysed flagella), This gene enco des a predicted protein of 788 amino acid residues and a molecular wei ght of 90977 with no significant homology to known proteins. Site-spec ific insertional mutants into this open reading frame result in the sa me 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 a dhesion, although other adhesins mediate a motility-dependent internal ization process. Characterization of the mutants at the molecular leve l and in animal models should further contribute to our understanding of the pathogenicity of these organisms.