MOLECULAR CHARACTERIZATION OF THE TIA INVASION LOCUS FROM ENTEROTOXIGENIC ESCHERICHIA-COLI

Enterotoxigenic Escherichia call (ETEC) shares with other diarrheal pa thogens the capacity to invade epithelial cell lines originating from the human ileum or colon, although the role of invasion in ETEC pathog enesis remains undefined. Two distinct loci (tia and tib) that direct noninvasive E. coli to adhere to and invade intestinal epithelial cell lines have previously been isolated from cosmid libraries of the clas sical ETEC strain H10407. Here, we report the molecular characterizati on of the tia locus. Sodium dodecyl sulfate-polyacrylamide gel electro phoresis analysis of cellular fractions of E. coli DH5 alpha carrying the tia-positive cosmids and recombinant plasmid subclones revealed th at this locus directs the production of a 25-kDa protein (the Tia prot ein) that is localized to the outer membrane. The tia locus was subclo ned to a maximum of 2 kb and mutagenized with bacteriophage Mud. Synth esis of this protein was directly correlated with the ability of subcl ones and Mud transposon mutants to adhere to and invade epithelial cel ls. Sequencing of the tia locus identified a 756-bp open reading frame . All transposon insertions resulting in an invasion-negative phenotyp e mapped to this open reading frame. The open reading frame was amplif ied and directionally cloned behind the inc promoter of pHG165. This c onstruct directed DH5 alpha to express a 25-kDa protein and to adhere to and invade epithelial cells. The role of the tia gene in directing epithelial adherence and invasion was further assessed by the construc tion of chromosomal tia deletion derivatives of the parent ETEC strain , H10407. These tia deletion strains were noninvasive and lacked the a bility to adhere to human ileocecal cells. The tia gene shares limited homology with the Yersinia ail locus and significant homology with th e hra1 agglutinin gene cloned from a porcine ETEC strain. Additionally , tia probes hybridized to geographically diverse ETEC strains, as wel l as some enteropathogenic E. coli, enteroaggregative E. coli, and Shi gella sonnei strains.