vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri

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Abstract
Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of Shigella flexneri 2a YSH6000 with reduced invasive capacity was isolated by Tn5 insertion mutagenesis. The linkage of the mutant phenotype to the Tn5 insertion was determined by P1 phage transduction. The site of the Tn5 insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2,280-bp open reading frame encoding an 86.9-kDa protein located 669 bp downstream from the 3' end of the open reading frame for the purA gene. Disruption of the vacB gene of other serotypes of Shigella spp. and enteroinvasive Escherichia coli (EIEC) resulted in reduced expression of virulence phenotypes, indicating that the vacB gene encodes a novel type of virulence-associated gene required for the full expression of the virulence phenotype of Shigella spp. and EIEC.