title: Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm.
abstract: 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.
authors.affiliation: Centro Ricerche ENEA-Casaccia, Divisione PRO-TOSS, S. Maria di Galeria, Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bartoleschi
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Pardini
authors.forename: Claudia
authors.initials: C
authors.lastname: Scaringi
authors.forename: Maria Celeste
authors.initials: MC
authors.lastname: Martino
authors.forename: Carlo
authors.initials: C
authors.lastname: Pazzani
authors.forename: Maria Lina
authors.initials: ML
authors.lastname: Bernardini
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002702:Chloramphenicol Resistance / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics / Q000382:microbiology* / Q000473:pathology*; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D012763:Shigella flexneri / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D010948:Viral Plaque Assay / Q000379:methods; D014774:Virulence / Q000235:genetics*