Chapter 12 : Transcytosis of Bacterial Toxins across Mucosal Barriers

Absorptive mechanisms of transporting epithelia. (A) Polarized epithelial cells lining mucosal surfaces form a barrier that prevents penetration of macromolecules and bacterial toxins beyond the epithelium. (B) Some molecules move between cells and pass through the tight junctions by the process of paracellular transport. (C) In contrast, small solutes such as sodium and glucose cross the epithelial barrier by the action of specific transporters and channels that facilitate their movement in a process termed transcellular transport.

Absorptive mechanisms of transporting epithelia. Direct transcytosis of dIgA-pIgR complexes (left) is initiated by binding of dIgA to the pIgR at the basolateral surface of the epithelial cell, which stimulates endocytosis of the complex into an early endosome (EE). Here, the dIgA-pIgR complex moves away from fluid-phase cargo and other membrane components targeted to the late endosome or lysosome for degradation. The dIgA-pIgR complex then traffics into the common endosome (CE) and then again to the apical recycling endosome (ARE). Transport from the common endosome into the apical recycling endosome is a hallmark of polarized sorting of the pIgR in the transcytotic pathway. In indirect transcytosis (right panel), CT enters the polarized epithelial cell by binding GM1 at the apical membrane and then moves via an early endosomal compartment (AE) into the Golgi complex. In the Golgi, the C-terminal KDEL motif on the A-subunit facilitates retrograde movement of the CT-GM1 complex through the Golgi stack and into the ER. In the ER, the A-subunit separates from the B-subunit-GM1 complex, unfolds, and translocates across from the ER into the cytosol in a process catalyzed by the ER lumenal chaperone protein disulfide isomerase (PDI). Alternatively, the A-subunit may remain attached to the B-subunit after translocation and moves to the basolateral membrane by entering the anterograde transport vesicles (BE) and moving back out the secretory pathway.

Immunolocalization of FcRn in polarized T84 monolayers and normal adult human small intestinal mucosa. (a) Whole-mount T84 monolayers visualized en face show a diffuse, punctate staining pattern. The Z0-1 image was captured slightly above the focal plane of FcRn. (b) FcRn staining of whole-mount T84 monolayers visualized as confocal vertical sections. (c) FcRn staining was absent in the presence of an isotype-matched, irrelevant antiserum. (d and e) FcRn staining was absent in the presence of an irrelevant antiserum or with secondary antibody alone. (f) Villous enterocytes of normal adult human small intestine show delicate linear staining in the region of the apical cytoplasmic membrane. (g) Crypt enterocytes show an apical and punctuate staining pattern visible not only at the level of the apical cytoplasmic membrane, but also in the apical cytoplasm below the level of the apical membrane. (See Color Plates following p. 256.)

Crystal structure of CT. A ribbon diagram based on the crystal structure of the CT holotoxin is shown viewed from the side. Each subunit is labeled. This figure was kindly provided by Ethan Merritt and Wim Hol, University of Washington, Seattle.

Transcytosis of CT B-subunit across polarized T84 cell monolayers and effect of mutation in KDEL on CT trafficking through the transcytotic pathway. Transcytosis of KDEL-mutant (m) and wild-type (wt) CT from apical to basolateral membranes was assessed by selective cell surface biotinylation. T84 cell monolayers grown on permeable supports were exposed continuously to 1 nM of KDEL-mutant or wt CT at the apical cell surface at the indicated temperatures and times followed by cell surface biotinylation at either the apical (lane 1) or basolateral (lanes 2–10) surface. The upper panel shows a Western blot to demonstrate that each lane contains equivalent amounts of immunoprecipitated CT B subunit. The lower panel shows the avidinhorseradish peroxidase (HRP) blot of the same experiment, revealing biotinylated toxin. The first lane shows that CT B subunit can be labeled with biotin while bound to GM1 at the apical plasma membrane (positive control). At 4°C, a temperature that completely inhibits vesicular traffic, CT B does not reach the basolateral membrane, as indicated by the absence of biotinylated CT B subunit (lane 2). Lanes 7 and 8 show that in cells incubated at 27°C for 160 min, basolaterally applied biotin has now labeled a fraction of the Bsubunit at the basolateral membrane, indicating that both the KDEL-mutant and wt CT reach the basolateral membrane by transcytosis. The amount of the KDEL-mutant CT reaching the basolateral membrane is approximately fivefold less than that of wt CT, indicating that transcytosis for the KDELmutant CT proceeds more slowly than for wt toxin. From reference 6 with permission.