Zonula Occludens Toxin Structure-Function Analysis

Abstract

Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.Vibrio cholerae produces a variety of extracellular products including zonula occludens toxin (Zot) 1 (1). The zot gene, along with other genes encoding virulence factors such as ctxA, ctxB (2, 3), and ace (4), is part of the chromosomally integrated genome of a filamentous phage designated CTX⌽ (5-10). The zot product seems to be involved in the CTX⌽ morphogenesis because Zot mutagenesis studies demonstrated the inability of CTX elements to be self-transmissible under appropriate conditions (5). The high concurrence among V. cholerae strains of the zot gene and the ctx genes (11, 12) also suggests a possible synergistic role of Zot in the causation of acute dehydrating diarrhea typical of cholera. The recently completed genomic sequence of V. choleare El Tor N16961 revealed that the CTX⌽ filamentous phage is integrated in one of the two circular chromosomes of the bacterium (13).Beside its role in phage morphogenesis, Zot also increases the permeability of the small intestine by affecting the structure of the intercellular tight junctions (tj) (1). This effect was initially described on rabbit ileal tissues mounted in Ussing chambers by using filtered supernatants from V. cholerae O1 strains, suggesting that Zot is secreted (1, 14). Zot also possesses a cell specificity related to the toxin interaction with a specific receptor whose surface expression differs on various cells (15-17). Zot induces modifications of cytoskeletal organization that lead to the opening of tj secondary to the transmembrane phospholipase C and subsequent protein kinase C␣-dependent polymerization of actin filaments strategically localized to regulate the paracellular pathway (15). Furthermore, in vivo experiments suggested that the effect of Zot on tj might lead to intestinal secretion after...

Role of the intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-prone rats

“…Experiments were carried out as we have described (17,19,23). Briefly, male BBDP and BBDR rats (age range, 20-110 days) were killed as described above.…”

“…In this study, we used the combination of the Ussing chamber assay and a recently developed zonulin sandwich ELISA to study whether zonulin was responsible for this early increase in gut permeability typical of BBDP rats (13). Furthermore, we used a synthetic peptide, competitive inhibitor (FZI͞0) (23) to confirm the role of zonulin in T1D pathogenesis and to possibly develop therapeutic and͞or preventive interventions for autoimmune diseases characterized by leaky gut.…”

Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses

“…Although the exact adjuvant mechanism of Zot is not known, it is conceivable that this effect is related to the ability of Zot to open tight junctions, with the subsequent increase in permeability of the mucosal epithelial barrier (9,11,12). It has been reported that Zot may mimic the endogenous molecule zonulin, with which it seems to share a receptor on intestinal epithelial cells (3,26). An octapeptide (FZI/0) homologous to the Zot-zonulin putative binding site that blocks the permeability effects of both Zot and zonulin has been synthesized (3,26).…”

“…It has been reported that Zot may mimic the endogenous molecule zonulin, with which it seems to share a receptor on intestinal epithelial cells (3,26). An octapeptide (FZI/0) homologous to the Zot-zonulin putative binding site that blocks the permeability effects of both Zot and zonulin has been synthesized (3,26). To understand whether the putative …”

“…It then binds to its receptor and modulates tight junctions (8). Interestingly, Zot and zonulin bind to the same receptor on intestinal epithelial cells and share a common binding motif at their N termini (3,26). Thus, it appears that Zot mimics a physiological molecule in the regulation of tight junctions, and this mechanism may be responsible for its mucosal adjuvant effect.…”