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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.

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A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plasmid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for 0-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.