Identification and molecular characterization of a 27 kDaShigella flexneriinvasion plasmid antigen, IpaJ

Abstract

Shigellaspecies and enteroinvasiveEscherichia colicontain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains ofShigellae.Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen fromShigella flexneri, designated IpaJ. TheipaJgene encodes a 780 bp open reading frame (ORF), separated from theipaR(virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoAmutagenesis of theipaJORF confirmed the presence of export signal sequences in IpaJ. UnlikeipaBCDAgenes, transcription analysis ofipaJindicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as aShigellaimmunogen. Construction and phenotypic characterization ofipaJmutants in two serotypes ofS. flexnerishowed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, theipaJmutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.