Zonula occludens toxin structure-function analysis - Identification of thefragment biologically active on tight junctions and of the zonulin receptor binding domain

Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholera e that increases intestinal permeability by interacting with a mammalian ce ll receptor with subsequent activation of intracellular signaling leading t o the disassembly of the intercellular tight junctions. Zot localizes in th e bacterial outer membrane of V. cholerae with subsequent cleavage and secr etion of a carboxyl-terminal fragment in the host intestinal milieu. To ide ntify the Zot domain(s) directly involved in the protein permeating effect, several tot gene deletion mutants were constructed and tested for their bi ological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologi cally active domain was localized toward the carboxyl terminus of the prote in and coincided with the predicted cleavage product generated by V. choler ae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid co mparison between the Zot active fragment and zonulin, combined with site-di rected mutagenesis experiments, confirmed the presence of an octapeptide re ceptor-binding domain toward the amino terminus of the processed Zot.