Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain.
Intimins, encoded by eae genes, are outer membrane proteins involved in attaching-effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium. A series of intimins, harbouring specific mutations close to the C-terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp-2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin-mediated A/E lesion formation can be segregated from intimin-mediated HEp-2 cell invasion. Moreover, adherence to IVOC, EPEC-induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
G
G
Frankel
A D
AD
Philips
M
M
Novakova
M
M
Batchelor
S
S
Hicks
G
G
Dougan
D000199:Actins / Q000032:analysis / Q000378:metabolism; D018829:Adhesins, Bacterial; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000382:microbiology; D002352:Carrier Proteins; D002448:Cell Adhesion; D002648:Child; D002675:Child, Preschool; D002954:Citrobacter / Q000235:genetics / Q000472:pathogenicity*; D003106:Colon / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005260:Female; D006801:Humans; D007223:Infant; D007422:Intestines / Q000382:microbiology*; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014407:Tumor Cells, Cultured
D000199:Actins; D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli