CHARACTERIZATION OF VIRULENCE GENES OF ENTEROINVASIVE ESCHERICHIA-COLI BY TNPHOA MUTAGENESIS - IDENTIFICATION OF INVX, A GENE REQUIRED FOR ENTRY INTO HEP-2 CELLS

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotyp ically nearly identical, a difference has been reported in the infecti ve dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasivenes s of these bacteria toward epithelial cells. Thus, despite the high de gree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may ex ist. We have undertaken a systematic genetic analysis of the EIEC plas mid pSF204, using transposon mutagenesis. Congo red-negative TnphoA in sertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were is olated and screened for the ability to invade cultured HEp-2 cells. Mo st invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mx i, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxi M, and mxiD homologs and in a proposed new gene, named invX, located d ownstream of the spa region were identified and characterized. This an alysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a stric t requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.