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Journal of Clinical Microbiology, December 1999, p. 3822-3827, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of a Universal Intimin Antiserum and PCR Primers

Miranda Batchelor,1 Stuart Knutton,2 Alfredo Caprioli,3 Veronika Huter,1,dagger Mazlina Zanial,1 Gordon Dougan,1 and Gad Frankel1,*

Department of Biochemistry Imperial College of Science, Technology and Medicine, London SW7 2AZ,1 and Institute of Child Health, University of Birmingham, Birmingham B4 6NH,2 United Kingdom, and Laboratorio di Medicina Veterinaria, Istituto Superiore di Sanita, Rome, Italy3

Received 7 May 1999/Returned for modification 24 June 1999/Accepted 23 August 1999

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) constitute a significant risk to human health worldwide. A hallmark of both pathogens is their ability to produce characteristic attaching-and-effacing (A/E) lesions in intestinal epithelial cells. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin, an LEE-encoded bacterial adhesion molecule, mediates the intimate bacterium-host cell interaction characteristic of A/E lesions. On the basis of characterization of the C-terminal 280-amino-acid cell binding domain of intimin (Int280661-939), four distinct Int280 types (types alpha , beta , gamma , and delta ) have been identified. Importantly, Int280alpha and Int280beta antisera specifically recognized their respective intimin types. Using a conserved region of the intimin molecule (Int388-667) and primers synthesized to generate the recombinant Int388-667, we have now generated universal intimin antiserum and PCR primers that are reactive with the different intimin types expressed by both human and animal A/E lesion-forming strains. Use of immunogold electron microscopy to visualize intimin on the surfaces of EPEC and EHEC strains revealed, in general, a uniform distribution on the bacterial cell surface. However, a filamentous staining pattern was observed with a few strains expressing intimin gamma . Cloning of the intimin eae gene from one such strain (strain ICC57) into strain CVD206, an EPEC strain which harbors a null deletion in eae, produced a uniform intimin staining pattern indicating that, if the filamentous staining pattern defines a filamentous form of intimin gamma , it is dependent upon the genetic background of the strain and is not a feature of the intimin molecule.

* Corresponding author. Mailing address: Department of Biochemistry, Imperial College, Exhibition Rd., London SW7 2AZ, United Kingdom. Phone: 44-71-594-5253. Fax: 44-71-594-5255. E-mail: g.frankel{at}ic.ac.uk.

dagger Present address: Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

Journal of Clinical Microbiology, December 1999, p. 3822-3827, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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