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. 1999 May 17;145(4):689-98.
doi: 10.1083/jcb.145.4.689.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells

W L Beatty  1 , S Méresse, P Gounon, J Davoust, J Mounier, P J Sansonetti, J P Gorvel
Affiliations

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells

W L Beatty et al. J Cell Biol. .

Abstract

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

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Figures

Figure 1
Figure 1
Kinetics of LPS released at the apical and basolateral surfaces of polarized epithelial cells. T84 cell monolayers were preloaded for 2 h with 2 μg/ml of LPS at the apical pole. After subsequent washing to remove extracellular LPS, the fate of the cell-associated LPS was analyzed at the times indicated. The results are expressed as the mean ± SD of three individual monolayers.
Figure 2
Figure 2
Analysis of LPS interaction with the apical surface of polarized T84 cells by electron microscopy. After internalization of LPS-coated colloidal gold at the apical plasma membrane, these particles (arrows) were found in association with coated pits (a–c). In addition, LPS-coated particles could be localized to coated vesicles at the apical surface (c). Bar = 200 nm.
Figure 3
Figure 3
Analysis of transcytosis of LPS by transmission electron microscopy. (a, e, and f) Cross-section of a polarized T84 cell monolayer, after internalization of LPS-coated gold particles at the apical pole. Panel a shows apical villi, tight junctions, inter(para)cellular space (i.s.), and basally positioned nucleus. LPS-coated particles primarily localize to vesicles in the apical region of the polarized cell (arrows), some of them in close proximity of the lateral region opposing the paracellular space. e and f show the intercellular region below the tight junctions, with LPS appearing within basolateral endosomes adjacent to the lateral membrane and proceeding to exocytosis into the paracellular space. (b–d) Double immunogold labeling of LPS (10 nm gold particles) and the transferrin receptor (5 nm gold particles). Panel b shows two adjacent vesicles, one containing the transferrin receptor alone (bottom) and one (top) containing both LPS and the transferrin receptor. Panels c and d show multimembranous vesicles containing both LPS and the transferrin receptor. Bars: (a) 500 nm; (b–f) 100 nm.
Figure 4
Figure 4
Apically internalized LPS localized to a basolateral transferrin-containing compartment. (A) LPS was internalized for 20 min from the apical domain of polarized T84 cells (column 2). Transferrin-FITC (Tf) was added to the basolateral pole for the last 10 min of internalization (column 1). Consecutive horizontal planes in a confocal series are shown representing the apical (2 μm from plasma membrane), medial (10 μm below the apical scan), and basal (2 μm above the filter) region of the epithelial monolayer. The corresponding images of each plane were superimposed in a double-color image in which LPS appears in red and the pattern of transferrin labeling is in green (column 3). Arrows indicate LPS and transferrin colocalization in the apical region of the cells, these structures being yellow in color when the images are superimposed (column 3). (B) LPS was added to the apical pole of polarized T84 cells for 20 min followed by incubation in LPS-free medium for an additional 40 min (column 2). Transferrin-FITC was added at the basolateral surface for the final 10 min of internalization (column 1). LPS remained in the apical region of the polarized cells, thus only the apical plane is shown. Superimposed images (column 3) revealed that colocalization between LPS and transferrin (as indicated by the arrow) declined with extended time of LPS internalization. Bar, 5 μm.
Figure 5
Figure 5
Fluid-phase markers followed an endocytic pathway distinct from LPS. (Top) BSA-rhodamine was internalized for 20 min from the apical domain of polarized T84 cells and transferrin-FITC (Tf) was added to the basolateral side of the epithelium for the last 10 min of internalization. Apical BSA did not colocalize with basolateral transferrin (arrowheads). (Bottom) LPS was added apically for 20 min and BSA-FITC was added basolaterally for the final 10 min of internalization. The majority of apical LPS did not colocalize with basolateral BSA (arrowheads). Arrows show limited colocalization between LPS and BSA. Analysis of cells resulted in localization of fluorescent molecules within the apical region of polarized cells, therefore only the scan corresponding to a plane 2 μm from the apical surface is shown. Column 3 illustrates the superimposed images. Bar, 5 μm.
Figure 6
Figure 6
Colocalization of apically internalized LPS with markers for late endosomes and lysosomes. LPS was internalized at the apical surface of T84 cells for 20 min followed by no chase (0) or a chase in LPS-free medium for 40 min (40′) or 1 h and 40 min (1h40′). (Top) Anti–CI-MPR antibody was used to detect localization of LPS to late endosomal compartments. (Bottom) Anti–Lamp 2 antibody was used to localize LPS to lysosomal compartments. Only the apical plane is shown. Superimposed images revealed that at 40 min chase, LPS strongly colocalized with CI-MPR. At 1 h and 40 min chase, LPS predominantly localized to lysosomal compartments. Bar, 5 μm.
Figure 7
Figure 7
Model for intracellular trafficking of apically internalized LPS in polarized T84 cells. Apical LPS is internalized and after 20 min reaches a LPS-recycling compartment (LPS-RC). LPS-RC contains transferrin (Tf) and is poorly accessible to apically (a-BSA) and basolaterally (b-BSA) internalized BSA accumulated in apical (a-EE) and basolateral (b-EE) early endosomes, respectively. 38% of internalized LPS are recycled to the apical reservoir, 15% transcytosed and 47% targeted to late endosome (LE)/ lysosomal (Lys) compartments.
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