Identification of regions on a 230-kilobase plasmid from enteroinvasive Escherichia coli that are required for entry into HEp-2 cells

Abstract

Certain strains of Escherichia coli can cause an invasive diarrheal disease in humans which clinically resembles shigellosis. These strains share with Shigella species the ability to enter and replicate within colonic epithelial cells and the ability to bind Congo red dye in vitro when grown at 37°C. Like shigellae, they contain a large plasmid essential for virulence. A 230-kilobase (kb) plasmid from enteroinvasive E. coli was genetically marked with a transposan and mobilized into an E. coli K-12 background. This plasmid conferred upon E. coli K-12 the ability to enter and multiply within cultured epithelial cells, as well as the ability to bind Congo red. Expression of these phenotypes required growth at 37°C. Transposon mutagenesis was used to identify regions on the 230-kb plasmid required for virulence. All transposon insertions which resulted in loss of the ability to enter epithelial cells, as wel as the ability to bind Congo red dye, were mapped to a single 25-kb BamHI fragment. Subclones from this 25-kb region were tested for the ability to complement invasion of noninvasive derivatives. A subclone containing about 8 kb of the left end of the 25-kb BamHI fragment was capable of complementing noninvasive mutants with Tn5 insertions in this region and restored to these noninvasive mutants the ability to enter epithelial cells.