Identification of Shigella invasion genes by isolation of temperature-regulated inv::lacZ operon fusions

Abstract

Penetration and multiplication within cells of the human colonic epithelium are hallmarks of Shigella spp. pathogenicity. Shigella spp. virulence is regulated by growth temperature. Strains phenotypically virulent when grown at 37°C are phenotypically avirulent when grown at 30°C. The number of genes involved in Shigella spp. pathogenicity and how many virulence genes are temperature regulated are unknown. To facilitate the study of temperature-regulated virulence in Shigella spp., we employed lacZ operon fusion technology to identify temperature-regulated invasion (inv) genes. Four inv::lacZ fusion mutants were identified and found to be unable to invade HeLa cells. The fusions were located in a region of the 220-kilobase invasion plasmid defined as the minimal amount of DNA required for invasion, and they were controlled by virR, the temperature-dependent virulence gene regulator. Western blot (immunoblot) and Southern hybridization analyses indicated that one of the fusions was located in a known inv gene, ipaB, which encodes one of the major immunogenic peptides of Shigella spp. This ipaB::lacZ operon fusion mutant synthesized a truncated IpaB protein recognized by IpaB-specific monoclonal antibodies. Three of the fusions were within novel genes mapping to regions previously identified as essential for a positive virulence phenotype. Analysis of bacterial surface proteins suggested that the genes marked by these fusions may play a role in the correct surface expression of the ipaB and ipaC gene products.