PROTEOLYTIC ACTIVATION OF CHOLERA-TOXIN AND ESCHERICHIA-COLI LABILE TOXIN BY ENTRY INTO HOST EPITHELIAL-CELLS - SIGNAL-TRANSDUCTION BY A PROTEASE-RESISTANT TOXIN VARIANT

Cholera and Escherichia coli heat-labile toxins (CT and LT) require pr oteolysis of a peptide loop connecting two major domains of their enzy matic A subunits for maximal activity (termed ''nicking''). To test wh ether host intestinal epithelial cells may supply the necessary protea se, recombinant rCT and rLT and a protease-resistant mutant CTR192H we re prepared, Toxin action was assessed as a Cl- secretory response (Is c)elicited from monolayers of polarized human epithelial T84 cells, Wh en applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 mu A/cm(2)). Ise was reduced a-fold, however, when toxins were applied to basolateral membranes, Pretreatment of wild ty po toxins with trypsin in vitro restored the ''basolateral'' secretory responses to ''apical'' levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a se rine-type protease; T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when a pplied to apical membranes. Thus, T84 cells express a serine-type prot ease(s) fully sufficient, for activating the A subunits of CT and LT, The protease, however, is only accessible for activation when the toxi n enters the cell via the apical membrane.