Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells

Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells. Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned. These studies resulted in the grouping of the mutants into five main categories. These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization. These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.

[1]  G. Keusch,et al.  Epithelial cell invasion: an overlooked property of enteropathogenic Escherichia coli (EPEC) associated with the EPEC adherence factor. , 1989, The Journal of infectious diseases.

[2]  J. I. Phillips,et al.  Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model , 1989, Infection and immunity.

[3]  R. Isberg,et al.  Determinants for thermoinducible cell binding and plasmid-encoded cellular penetration detected in the absence of the Yersinia pseudotuberculosis invasin protein , 1989, Infection and immunity.

[4]  J. Mekalanos,et al.  Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae , 1989, Journal of bacteriology.

[5]  P. Williams,et al.  Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli , 1989, Infection and immunity.

[6]  J. R. Andrade,et al.  An endocytic process in HEp-2 cells induced by enteropathogenic Escherichia coli. , 1989, Journal of medical microbiology.

[7]  G. Dougan,et al.  Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer , 1988, Molecular microbiology.

[8]  M. Skurnik,et al.  Increased virulence of Yersinia pseudotuberculosis by two independent mutations , 1988, Nature.

[9]  S. Falkow,et al.  Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells , 1988, Infection and immunity.

[10]  P. Sansonetti,et al.  Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Falkow,et al.  Identification of invasin: A protein that allows enteric bacteria to penetrate cultured mammalian cells , 1987, Cell.

[12]  S. Falkow,et al.  Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells , 1987, Infection and immunity.

[13]  S. Knutton,et al.  Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa , 1987, Infection and immunity.

[14]  J. Kaper,et al.  Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells , 1987, Infection and immunity.

[15]  R. Robins-Browne.  Traditional enteropathogenic Escherichia coli of infantile diarrhea. , 1987, Reviews of infectious diseases.

[16]  M. Levine,et al.  Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. , 1985, The Journal of infectious diseases.

[17]  S. Falkow,et al.  A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12 , 1985, Nature.

[18]  M. Levine,et al.  The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. , 1985, The Journal of infectious diseases.

[19]  S. Tzipori,et al.  Enteropathogenic Escherichia coli enteritis: evaluation of the gnotobiotic piglet as a model of human infection. , 1985, Gut.

[20]  L. Trabulsi,et al.  Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells , 1984, Infection and immunity.

[21]  M. Levine,et al.  Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines , 1983, Infection and immunity.

[22]  M. Levine,et al.  Plasmid‐Mediated Adhesion in Enteropathogenic Escherichia coli , 1983, Journal of pediatric gastroenterology and nutrition.

[23]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[24]  M. Levine,et al.  Summary of a workshop on enteropathogenic Escherichia coli. , 1983 .

[25]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[26]  K. Otto,et al.  pUR222, a vector for cloning and rapid chemical sequencing of DNA. , 1981, Nucleic acids research.

[27]  M. Ulshen,et al.  Pathogenesis of escherichia coli gastroenteritis in man--another mechanism. , 1980, The New England journal of medicine.

[28]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[29]  Daniel S Waterman,et al.  ESCHERICHIA COLI STRAINS THAT CAUSE DIARRHŒA BUT DO NOT PRODUCE HEAT-LABILE OR HEAT-STABLE ENTEROTOXINS AND ARE NON-INVASIVE , 1978, The Lancet.

[30]  L. Emödy,et al.  Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea. , 1977, Acta microbiologica Academiae Scientiarum Hungaricae.

[31]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[32]  L. Corley,et al.  Attachment and penetration of Escherichia coli into intestinal epithelium of the ileum in newborn pigs. , 1969, The American journal of pathology.