Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

Abstract

Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide in vitro and in vivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7 + vesicles and in basal exocytic Rab11 + vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular redistribution that coincides with the reorganization of COPI + and COPII + vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals. One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis

“…The timing of LMAN1 redistribution to the basal compartment coincides with the presence of hIL-10 being detected in nonpolarized cells within the lamina propria, where it was not associated with LMAN1. This redistribution of LMAN1 associated with AMT-101 A→B transcytosis is identical to that previously observed for Chx (19).…”

“…We next wanted to ensure that the genetic construction of AMT-101 retained the A→B transcytosis properties that should be imparted by Chx, which was previously shown to undergo receptor-mediated uptake and vesicular trafficking without targeting to the lysosomal degradation pathway in polarized enterocytes (19). Chx386 and hIL-10 elements of the fusion protein AMT-101 remained together during A→B transcytosis and within the lamina propria, allowing localization of hIL-10 to accurately describe the distribution of AMT-101 (Supplemental Fig.…”

“…Lectin, mannose binding 1 (LMAN1) protein, a resident of the endoplasmic reticulum-Golgi intermediate compartment and known to be involved in the sorting or recycling of proteins and lipids, is involved in Chx transcytosis (19). AMT-101 also colocalized with LMAN1 that redistributes within enterocytes during A→B transcytosis (Supplemental Fig.…”

“…The A→B transcytosis pathway accessed by Chx has been shown to intersect with Rab7 + and LAMP1 + vesicles; although these are markers of both late endosomes and lysosomes, Chx does not appear to traffic to lysosome-like structures within enterocytes (19). To examine this point for AMT-101, a time course of LAMP1 and AMT-101 colocalization was performed (Supplemental Fig.…”

“…The proximal half of Chx hijacks host cell vesicular trafficking elements to facilitate the efficient transcytosis, delivering intact toxin to cells within the underlying lamina propria; a different trafficking mechanism occurs within the lamina propria that allows the latter half of Chx to escape vesicular structures to reach the cytoplasm of these nonpolarized cells, where it enzymatically blocks protein synthesis (18). We have recently demonstrated that the proximal portion of Chx is capable of efficiently delivering a conjoined protein that replaces the latter (toxic) half of the protein to the intestinal lamina propria following oral administration (19). To focus an IL-10 payload to the intestinal lamina propria, a genetic fusion gene of Chx386 and hIL-10 with a glycine-serine-type linker was prepared (20).…”