Down regulation of intimin expression during attaching and effacing enteropathogenic Escherichia coli adhesion.
Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.
Knutton
S
S
Institute of Child Health, University of Birmingham, United Kingdom. S.KNUTTON@bham.ac.uk
Adu-Bobie
J
J
Bain
C
C
Phillips
A D
AD
Dougan
G
G
Frankel
G
G
D000199:Actins / Q000276:immunology; D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial / Q000276:immunology; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002352:Carrier Proteins; D002478:Cells, Cultured; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D019084:Fluorescent Antibody Technique, Indirect; D016253:Microscopy, Immunoelectron; D010957:Plasmids / Q000235:genetics; D014774:Virulence
D000199:Actins; D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli