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Infection and Immunity, April 2006, p. 2233-2244, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2233-2244.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

Alison S. Low,1 Francis Dziva,2 Alfredo G. Torres,3 Jessenya L. Martinez,3 Tracy Rosser,1 Stuart Naylor,4 Kevin Spears,1 Nicola Holden,1 Arvind Mahajan,1,5 John Findlay,6 Jill Sales,8 David G. E. Smith,5,7 J. Christopher Low,4 Mark P. Stevens,2 and David L. Gally1*

Zoonotic and Animal Pathogens Research Laboratory, Centre for Infectious Diseases, Chancellor's Building, University of Edinburgh, Edinburgh, EH16 4SB, United Kingdom,1 Division of Microbiology, Institute for Animal Health, Compton Laboratory, Compton, RG20 7NN, United Kingdom,2 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1070,3 Animal Research, Scottish Agricultural College, Sir Stephen Watson Building, Bush Estate, Penicuik, Midlothian, United Kingdom,4 Moredun Research Institute, Pentland Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, United Kingdom,5 Institute of Molecular Plant Sciences, University of Edinburgh, Daniel Rutherford Building, Kings Buildings, Mayfield Road, Edinburgh, EH9 3JH, United Kingdom,6 Institute for Comparative Medicine, University of Glasgow Veterinary School, Glasgow, G61 1QH, United Kingdom,7 Biomathematics & Statistics Scotland, Edinburgh, EH9 3JZ, United Kingdom8

Received 29 July 2005/ Returned for modification 9 September 2005/ Accepted 9 January 2006

Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

* Corresponding author. Mailing address: Zoonotic and Animal Pathogens Research Laboratory, Centre for Infectious Diseases, Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom. Phone: 0131 2429379. Fax: 0131 2429385. E-mail: dgally{at}ed.ac.uk.

Editor: V. J. DiRita

Infection and Immunity, April 2006, p. 2233-2244, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2233-2244.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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