Development of a universal intimin antiserum and PCR primers.
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 (Int280(661-939)), 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 (Int(388-667)) and primers synthesized to generate the recombinant Int(388-667), 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.
Batchelor
M
M
Department of Biochemistry Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom.
Knutton
S
S
Caprioli
A
A
Huter
V
V
Zanial
M
M
Dougan
G
G
Frankel
G
G
D018829:Adhesins, Bacterial; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D016133:Polymerase Chain Reaction
D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D007106:Immune Sera; C073819:eaeA protein, E coli