The Large Virulence Plasmid of Shigella flexneri

All virulent strains of Shigella flexneri possess a large 220kb plasmid that mediates its virulence properties. This so-called the invasion plasmid has been shown to encode the genes for several aspects of Shigella virulence, including

- Adhesins that are involved in the adherence of bacteria onto the surface of target epithelial cells

- The production of invasion plasmid antigens (Ipa) that have a direct role in the Shigella invasion process

- Transport or processing functions that ensure the correct surface expression of the Ipa proteins

- The induction of endocytic uptake of bacteria and disruption of endocytic vacuoles

- The intra- and inter-cellular spreading phenotypes

- The regulation of plasmid-encoded vir genes

The presence of this plasmid was discovered in the 1980s, after the observation that essentially the entire chromosome of S. flexneri could be transferred to E. coli without reconstituting the virulence phenotype of the donor. However, the ability to invade tissue culture cells was transferred to E. coli by the conjugal mobilization of this plasmid from S. flexneri.  (see below)

The invasion locus on the virulence plasmid of Shigella is a pathogenicity island-like cluster that consists of 38 ORFs of the ipa-mxi-spa operons within a stretch of 32 kb of the plasmid. Genes within this locus are critical for Shigella invasion of mammalian cells, although certain genes outside this region are required for optimal invasion of tissue culture cells.

Evolution of the Shigella virulence plasmid
Recent genetic analyses suggest that shigellae do not constitute a distinct genus as traditionally believed but rather are within the genus of E. coli, much like the enteric pathogenic E. coli. These analyses indicate that Shigella emerged from E. coli seven or eight independent times during evolution, leading to three clusters of Shigella, each of which contains serotypes from multiple traditional species, and four or five additional forms, each of which contains one traditional serotype. The three main Shigella clusters are estimated to have evolved 35,000 to 270,000 years ago, which predates the development of agriculture and makes shigellosis one of the early infectious diseases of humans.

The defining event each time Shigella arose was almost certainly the acquisition of an historical precursor of the current-day virulence plasmid. The data also suggest that the loss of specific catabolic pathways (inability to utilize lactose and mucate and to decarboxylate lysine), loss of motility, and expansion of O-antigen diversity that are characteristic of Shigella strains occurred more recently than the acquisition of the plasmid.

Since the plasmid was acquired at distinct times, one would predict that differences reflecting the evolution of the plasmid could be obtained by genetic comparison of virulence plasmids of the seven different Shigella evolutionary groups. Subsequent to the acquisition of the virulence plasmid, divergence of Shigella clones from E. coli would involve clonal divergence (accumulation of mutations by base substitution), horizontal transfer of genetic material from other species, and loss of gene sequences that interfere with pathogenicity.

Certain horizontal gene transfer events have been key to the evolution of Shigella. A quintessential feature of Shigella is its ability to invade mammalian cells and access the cell cytoplasm, defining a niche unique among enteric Gram-negative bacteria, with the exception of enteroinvasive E. coli. Thus, the acquisition and evolution of the ipa-mxi-spa pathogenicity island, which encodes all of the genes required for cell invasion and phagolysosomal lysis, permitted a major alteration in pathogenesis. Likewise, the acquisition of virG (icsA), which mediates actin assembly on Shigella, and virF and virB, the regulators of the virG and ipa-mxi-spa loci, were key to the emergence of Shigella. Since all Shigella serotypes contain these loci, they were probably all present on the prototypic virulence plasmid.