Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm

Cell Microbiol. 2002 Sep;4(9):613-26. doi: 10.1046/j.1462-5822.2002.00216.x.


We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Chloramphenicol Resistance / genetics
  • Cytoplasm / genetics
  • Cytoplasm / microbiology*
  • Cytoplasm / pathology*
  • Gene Transfer Techniques
  • Genes, Bacterial*
  • Genes, Reporter
  • HeLa Cells / microbiology
  • Humans
  • Mice
  • Plasmids / genetics
  • Plasmids / isolation & purification
  • Promoter Regions, Genetic
  • Shigella flexneri / genetics*
  • Shigella flexneri / growth & development
  • Shigella flexneri / isolation & purification*
  • Viral Plaque Assay / methods
  • Virulence / genetics*