Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Abstract

Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl ؊ secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 A/cm 2 ). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type 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 serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

“…Moreover studies with human intestinal T84 cell lines have demonstrated that the presence of an unidentified host epithelial serine protease(s) is sufficient to process CT A (85). In light of these findings, it is very likely that host proteases are responsible for CT A activation during intestinal colonization of V. cholerae; however, there may be conditions, yet to be determined, during which Vibrio proteases such as VesA and HapA play significant roles in the processing and activation of CT A.…”

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

“…By contrast, Etx from enterotoxinogenic E. coli, as well as recombinant Ctx produced in E. coli, are normally isolated with their A-subunits intact; trypsin or other gut-associated proteases have been postulated to accomplish toxin activation in such cases (33). Recent studies on T84 cells have shown that a serine protease, which efficiently nicks and activates both CtxA and EtxA, is present on either the apical surface or in apicallyderived transport vesicles (31). In this respect, no difference was found in toxicity of commercial (nicked) preparations of Ctx and recombinant Ctx that was either unnicked or nicked with trypsin.…”

“…For Ctx and Etx to exhibit full toxicity, the A-subunits must undergo proteolytic cleavage or "nicking" at Arg-192 to give separate A1-and A2-fragments (31). In the case of cholera toxin, extracellular proteases, such as HA protease produced by V. cholerae, can efficiently nick and activate the A-subunit (32).…”

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

“…Receptor binding is followed by internalization of LT into vesicles which are retrograde transported to the Golgi apparatus and endoplasmic reticulum (Rappuoli et al, 1999). Proteolytic cleavage of the A subunit into A 1 and A 2 domains (Lencer et al, 1997) occurs and is followed by the translocation of the A 1 domain into the cytosol. The A 1 domain ADPribosylates the -unit of the G s protein, the G protein regulating the activity of adenylate cyclase, resulting in the permanent activation of adenylate cyclase (Rappuoli et al, 1999) which in turn causes abnormal increases in cyclic AMP (cAMP) levels (Nataro et al, 1998).…”