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Infection and Immunity, July 2001, p. 4257-4267, Vol. 69, No. 7
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.7.4257-4267.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Motility and the Polar Flagellum Are Required for Aeromonas caviae Adherence to HEp-2 Cells

Ali A. Rabaan,1 Ioannis Gryllos,1,dagger Juan M. Tomás,2 and Jonathan G. Shaw1,*

Division of Molecular and Genetic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom,1 and Departamento Microbiología, Facultad Biología, Universidad Barcelona, Diagonal 645, 08071 Barcelona, Spain2

Received 27 September 2000/Returned for modification 15 January 2001/Accepted 19 March 2001

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.

* Corresponding author. Mailing address: Division of Molecular and Genetic Medicine, Floor F, University of Sheffield Medical School, Beach Hill Rd., Sheffield S10 2RX, United Kingdom. Phone: 44-114-2713517. Fax: 44-114-2739926. E-mail: j.g.shaw{at}sheffield.ac.uk.

dagger Present address: Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.

Infection and Immunity, July 2001, p. 4257-4267, Vol. 69, No. 7
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.7.4257-4267.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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