Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells

Aeromonas caviae is increasingly being recognized as a cause of gastroenter itis, especially among the young. The adherence of aeromonads to human epit helial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of A, caviae to HEp-2 cells was significantly reduced by ei ther pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella, To precisely def ine the role of the polar flagellum in aeromonad adherence, we isolated the A. caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH, and flaJ Each gene was inactivated us ing a kanamycin resistance cartridge that ensures the transcription of down stream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing , mutant analysis, and Western blotting demonstrated that A. caviae has a c omplex flagellum filament composed of two flagellin subunits encoded by fla A and flaB, The predicted molecular mass of both flagellins was similar to 31,700 Ha; however, their molecular mass estimated by sodium dodecyl sulfat e-polyacrylamide gel electrophoresis was similar to 35,500 Ha, This aberran t migration was thought to be due to their glycosylation, since the protein s were reactive in glycosyl group detection assays. Single mutations in eit her flaA or flaB did not result in loss of flagella but did result in decre ased motility and adherence by approximately 50%. Mutation of flaH,flaJ, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence, However, mutation of flaG did not affect motilit y but did significantly reduce the level of adherence. Centrifugation of th e flagellate mutants (flaA, flaB, and flaG) onto the cell monolayers did no t increase adherence, whereas centrifugation of the aflagellate mutants (fl aH, fIaJ, and flaA flaB) increased adherence slightly. We conclude that max imum adherence of A. caviae to human epithelial cells in vitro requires mot ility and optimal flagellar function.