Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri

Abstract

A large plasmid is found in virulent isolates of Shigella sp. and encodes functions essential for invasion of mammalian cells. To identify plasmid sequences necessary for invasion, we isolated a series of Tn5 insertions in pWR100, the virulence plasmid of Shigella flexneri serotype 5. These insertions demonstrated that three separate EcoRI fragments of pWRlO0 were required for invasion of HeLa cells. However, the corresponding native EcoRI fragments, when cloned into pBR325, did not restore virulence to plasmidless strains. Construction of a A-sensitive, plasmidless Shigella recipient enabled us to shotgun clone plasmid DNA directly into S. flexneri by using the cosmid vector pJB8 and score for expression of invasive functions. In this fashion, we succeeded in isolating six independent recombinants which restored invasion of HeLa cells in plasmidless Shigella recipients. The cloned inserts all contained a common core of ca. 37 kilobases, thus defining a minimum sequence necessary for invasion of HeLa cells. Virulence-associated peptides produced by wild-type S. flexneri were also produced by the recombinants. Expression of these peptides and expression of invasiveness by the clones were regulated by growth temperature, as is expression of these traits in wild-type S. flexneri. A complete invasive phenotype was not expressed by the recombinants in that they failed to produce a positive Sereny test. Possible explanations for this behavior as it relates to the mechanism of bacterial invasion are discussed.

Galectin-3, a marker for vacuole lysis by invasive pathogens

“…Effectors encoded by theTTSS of Shigella are required both for the process of invasion and vacuole lysis (Maurelli et al, 1985;Page et al, 1999;Schuch et al, 1999). We thus wondered whether it was invasion or vacuole lysis step in which the TTSS activation was required for acquiring the G3CS.…”

Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins

“…Overlapping cosmids representing the entire virulence plasmid previously were constructed by inserting 40 kb fragments of pWR100 into the vector pJB8 (Maurelli et al, 1985).…”

“…Genes involved in both steps are carried on a 200 kb virulence plasmid (reviewed by Hale, 1991;Parsot, 1994). A 31 kb fragment of this plasmid is necessary and apparently sufficient for entry into epithelial cells (Maurelli et al, 1985;Sasakawa et al, 1988). This fragment is organized in two divergently transcribed regions which schematically encode secreted proteins, the IpaA-D proteins and a type III secretion system, the Mxi-Spa secretion apparatus.…”

A tale of two bacterial enteropathogens and one multivalent vaccine

“…Formal and Dennis Kopecko in the Walter Reed Army Institute of Research in Washington, DC, that identified the virulence plasmid as responsible for host cell invasion capacity, the distinguishing pathogenic property of Shigella (Sansonetti, Kopecko, & Formal, 1981;Sansonetti, D'Hauteville, Formal, & Toucas, 1982;Sansonetti, Kopecko, & Formal, 1982;Sansonetti et al, 1983). Sansonetti et al proceeded to characterise key regions of this plasmid that harboured genes encoding invasion capability, secreted effectors, and a unique cell-to-cell spread phenotype that utilised polymerisation of host cell actin (Baudry, Maurelli, Clerc, Sadoff, & Sansonetti, 1987;Bernardini, Mounier, D'Hauteville, Coquis-Rondon, & Sansonetti, 1989;Hale, Sansonetti, Schad, Austin, & Formal, 1983;Maurelli, Baudry, D'Hauteville, Hale, & Sansonetti, 1985).…”