Abstract
Aeromonas caviae
is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial 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 either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define 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 using a kanamycin resistance cartridge that ensures the transcription of downstream 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 complex flagellum filament composed of two flagellin subunits encoded by
flaA
and
flaB
. The predicted molecular mass of both flagellins was ∼31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was ∼35,500 Da. This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays. Single mutations in either
flaA
or
flaB
did not result in loss of flagella but did result in decreased 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 motility but did significantly reduce the level of adherence. Centrifugation of the flagellate mutants (
flaA, flaB
, and
flaG
) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (
flaH, flaJ
, and
flaA flaB
) increased adherence slightly. We conclude that maximum adherence of
A. caviae
to human epithelial cells in vitro requires motility and optimal flagellar function.