Penetration of Human Intestinal Epithelial Cells by Salmonella: Molecular Cloning and Expression of Salmonella typhi Invasion Determinants in Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-07, Vol.86 (13), p.5173-5177
• Main Authors: Elsinghorst, Eric A., Baron, Louis S., Kopecko, Dennis J.
• Format: Article
• Language: English
• Subjects: Bacteria; Bacteriology; Biological and medical sciences; Cell Line; Cloning, Molecular; Cosmids; Cytochalasin B - pharmacology; DNA; Epithelial cells; Epithelium - microbiology; Epithelium - ultrastructure; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic loci; Humans; Intestines - microbiology; Libraries; Microbiology; Microscopy, Electron; Mutation; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Phenotypes; Plasmids; Restriction Mapping; Salmonella; Salmonella typhi - drug effects; Salmonella typhi - genetics; Salmonella typhi - pathogenicity; Yersinia
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.13.5173

Salmonella typhi, the causative agent of typhoid fever, must invade the human gastrointestinal tract and multiply within the host to cause disease. We have cloned from S. typhi Ty2 a chromosomal region that confers upon Escherichia coli HB101 the ability to invade cultured human intestinal epithelial cells. Three invasion-positive recombinant cosmids were isolated and restriction endonuclease analyses of the inserts showed a 33-kilobase region of identity. Transmission electron microscopy of epithelial cells invaded by S. typhi Ty2 or E. coli HB101 carrying an invasion cosmid showed intracellular bacteria contained within endocytic vacuoles. One of the invasion cosmids was mutagenized with transposon Tn5 to identify the cloned sequences that are required for the invasive phenotype. Seven of 92 independent Tn5 insertions within the common 33-kilobase region eliminated invasive ability and revealed at least four separate loci that are required for invasion. Penetration of epithelial cells by Ty2 and HB101 carrying the cloned invasion determinants was inhibited by cytochalasin B and D, indicating that epithelial cell endocytosis of S. typhi is a microfilament-dependent event. The invasion cosmids were found to carry the recA and srlC genes indicating that the cloned invasion determinants are located at about 58 minutes on the S. typhi chromosome. With a segment of the cloned S. typhi invasion region used as a probe, homologous sequences were isolated from Salmonella typhimurium. Two independent S. typhimurium recombinant cosmids containing the co 33-kilobase common region identified in S. typhi were isolated, but these cosmids did not confer upon HB101 the ability to invade epithelial cells.