Use of a novel approach, termed island probing, identifies the Shigella flexneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins

Abstract

The she gene of Shigella flexneri 2a, which also harbors the internal enterotoxin genes set1A and set1B (F. R. Noriega, GenBank accession no. U35656, 1995) encodes a homolog of the virulence-related immunoglobulin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA, and Hap, from an avian pathogenic Escherichia coli, an enteropathogenic E. coli, S. flexneri 5, and Haemophilus influenzae, respectively. To investigate the possibility that this locus was carried on a larger deletable element, the S. flexneri 2a YSH6000T she gene was insertionally disrupted by allelic exchange using a Tn10-derived tetAR(B) cassette. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracycline-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysis confirmed loss of the she::tetAR(B) locus in six independent tetracycline-sensitive isolates. Sample sequencing over a 25-kb region flanking she identified four insertion sequence-like elements, the group II intron-like sequence Sf.IntA, and the 3' end of a second IgA protease-like homolog, sigA, lying 3.6 kb downstream and in an orientation inverted with respect to she. The deletion was mapped to chromosomal NotI fragment A and determined to have a size of 51 kb. Hybridization with flanking probes confirmed that at least 17.7 kb of the 51-kb deletable element was unique to the seven she+ strains investigated, supporting the conclusion that she lay within a large pathogenicity island. The method described in this study, termed island probing, provides a useful tool to further the study of pathogenicity islands in general. Importantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.