P. aeruginosa adhesion to the AP surface is N-glycan dependent. (A and B) Addition of Man or Glc, but not Gal, to the growth medium increases the level of Man on the cell surface. MDCK, ConA^r, and Calu-3 cells were cultured in the presence or absence of the indicated sugar. Shown are well-polarized monolayers grown on Transwells that were fixed without permeabilization and stained at either the AP or BL surface with FITC-ConA, which binds to Man in N-glycan chains. ConA^r cells have less Man on the AP and BL surfaces than do wt MDCK cells, but the addition of Man or Glc restores FITC-ConA binding to wt levels. The addition of Man or Glc enhances FITC-ConA binding to MDCK and Calu-3 cells at both the AP and BL surfaces. (A) 3D reconstructions of z-stack images acquired by confocal microscopy. Shown are AP and BL projections of well-polarized monolayers. Man is stained with FITC-ConA (green), and actin is stained with phalloidin (red). (B) FITC-ConA fluorescence (in arbitrary units) of well-polarized cells treated with the indicated sugars, as measured in a fluorescence plate reader. Shown are the means ± SD for three separate experiments. *, P < 0.05 compared to AP-side-untreated MDCK cells; ×, P < 0.05 compared to AP-side-untreated ConA^r cells; +, P < 0.05 compared to AP-side-untreated Calu-3 cells; **, P < 0.05 compared to BL-side-untreated MDCK cells; ××, P < 0.05 compared to BL-side-untreated ConA^r cells; ++, P < 0.05 compared to BL-side-untreated Calu-3 cells. (C and D) Addition of Man or Glc is sufficient to enhance P. aeruginosa binding. Cells were grown in the presence or absence of the indicated sugar, bacteria were added for 1 h to the AP or BL surface of well-polarized cells grown on Transwell filters, and standard adhesion assays were performed. Shown are the means ± SD for three separate experiments. (C) P. aeruginosa binding to well-polarized MDCK and ConA^r cells, normalized to AP-side-infected, untreated MDCK cells. (D) P. aeruginosa binding to well-polarized Calu-3 cells, normalized to AP-side-infected, untreated Calu-3 cells. (E and F) Tunicamycin inhibits bacterial binding to the AP surface of wt MDCK and Calu-3 cells in a dose-dependent manner. Cells were pretreated with the indicated amounts of tunicamycin to inhibit N-glycosylation. Bacterial binding was measured as described above. (E) P. aeruginosa binding to well-polarized MDCK and ConA^r cells, normalized to AP-side-infected, untreated MDCK cells (point “0”). (F) P. aeruginosa binding to well-polarized Calu-3 cells, normalized to AP-side-infected, untreated Calu-3 cells (point “0”). *, P < 0.05 compared to AP-side-infected, untreated MDCK cells; ×, P < 0.05 compared to AP-side-infected, untreated ConA^r cells; +, P < 0.05 compared to AP-side-infected, untreated Calu-3 cells.

HSPGs are expressed on the AP surface in incompletely polarized cells. In well-polarized monolayers grown on Transwell filters, there was a greater amount of HSPGs on the BL surface than on the AP surface. In incompletely polarized monolayers, there was increased expression of HSPGs on the AP surface. (A) 3D reconstruction of z-stack images acquired by confocal microscopy. Shown are AP and BL projections of well-polarized and incompletely polarized Calu-3 cells stained with an antibody against the HS chain of HSPGs (FITC-HS; purple), with phalloidin (red) to visualize actin, and with ZO-1 (green) to visualize tight junctions. (B and C) FITC-HS fluorescence (in arbitrary units) measured in a fluorescence plate reader. Well-polarized (B) or incompletely polarized (C) MDCK, ConA^r, and Calu-3 cells were stained with FITC-HS added to the AP or BL surface. Shown are the means ± SD for three separate experiments. *, P < 0.05 compared to AP surface-stained MDCK cells; ×, P < 0.05 compared to AP surface-stained ConA^r cells; +, P < 0.05 compared to AP surface-stained Calu-3 cells.

HS chains mediate P. aeruginosa adhesion to the BL surface and contribute to binding to the AP surface in incompletely polarized cells. Competitive inhibition with heparin (A to D) or enzymatic removal of HS chains with heparinase III (E to H) inhibits P. aeruginosa binding to the BL surface of well-polarized and incompletely polarized cells and to the AP surface of incompletely polarized cells. Cells were pretreated with the indicated amount of heparin or heparinase III, bacteria were added for 1 h, and standard adhesion assays were performed. Shown are the means ± SD for three separate experiments. P. aeruginosa binding to well-polarized (A) and incompletely polarized (B) MDCK and ConA^r cells pretreated with increasing amounts of heparin was determined and normalized to that of AP-side-infected, untreated MDCK cells. P. aeruginosa binding to well-polarized (C) and incompletely polarized (D) Calu-3 cells pretreated with increasing amounts of heparin was determined and normalized to that of AP-side-infected, untreated Calu-3 cells. P. aeruginosa binding to well-polarized (E) and incompletely polarized (F) MDCK and ConA^r cells pretreated with increasing doses of heparinase III was determined and normalized to that of AP-side-infected, untreated MDCK cells. P. aeruginosa binding to well-polarized (G) and incompletely polarized (H) Calu-3 cells pretreated with increasing doses of heparinase III was determined and normalized to that of AP-side-infected, untreated Calu-3 cells. *, P < 0.05 compared to AP-side-infected, untreated MDCK cells; ×, P < 0.05 compared to AP-side-infected, untreated ConA^r cells; +, P < 0.05 compared to AP-side-infected, untreated Calu-3 cells; **, P < 0.05 compared to BL-side-infected, untreated MDCK cells; ××, P < 0.05 compared to BL-side-infected, untreated ConA^r cells; ++, P < 0.05 compared to BL-side-infected, untreated Calu-3 cells.

P. aeruginosa binding to HS is dependent on sulfation of HS chains. Chemical inhibition of sulfation inhibits P. aeruginosa binding to the BL surface of well-polarized and incompletely polarized cells and to the AP surface of incompletely polarized cells. Cells were pretreated with sodium chlorate for desulfation (desulf) of HS chains, and sulfation was restored (resulf) by treatment with sodium sulfate. Bacteria were added for 1 h, and standard adhesion assays were performed. Shown are the means ± SD for three separate experiments. P. aeruginosa binding to well-polarized (A) and incompletely polarized (B) MDCK and ConA^r cells was determined and normalized to that of AP-side-infected, untreated MDCK cells. P. aeruginosa binding to well-polarized (C) and incompletely polarized (D) Calu-3 cells was determined and normalized to AP-side-infected, untreated Calu-3 cells. *, P < 0.05 compared to AP-side-infected, untreated MDCK cells; ×, P < 0.05 compared to AP-side-infected, untreated ConA^r cells; +, P < 0.05 compared to AP-side-infected, untreated Calu-3 cells; **, P < 0.05 compared to BL-side-infected, untreated MDCK cells; ××, P < 0.05 compared to BL-side-infected, untreated ConA^r cells; ++, P < 0.05 compared to BL-side-infected, untreated Calu-3 cells.

P. aeruginosa directly binds in vitro to isolated HS and N-glycan chains in a dose-dependent manner. Ninety-six-well plastic plates were coated overnight with increasing concentrations of the indicated molecules. GFP-conjugated bacteria were added for 1 h, and fluorescence was quantified in a fluorescence plate reader. Control bacterial binding to noncoated wells was set as a background, and the percentage of binding above that of the control is indicated. Shown are means ± SD for four separate experiments. HS, heparan sulfate; CS-4, 4-0-sulfated chondroitin sulfate; CS-6, 6-0-sulfated chondroitin sulfate; HA, hyaluronic acid; GlcN, N-acetylglucosamine.

Bacterial cytotoxicity is mediated upon P. aeruginosa binding to N-glycans on the AP surface of polarized cells, to HSPGs on the BL surface of polarized cells, and to both molecules on the AP surface of incompletely polarized cells. Cells were pretreated with mannose (man) to upregulate N-glycosylation, with tunicamycin (tun) (32) to inhibit N-glycosylation, with heparinase III (hepIII) to remove HS chains, with sodium chlorate for desulfation (desulf), or with sodium sulfate for resulfation (resulf). Host cell treatments are color coded as follows: modifications of HSPGs are shown in shades of red and modifications of N-glycans are shown in shades of blue. Bacteria were added for 5 h, and standard LDH release assays were performed. Shown are the means ± SD for four separate experiments. Percentages of LDH release in well-polarized (A) and incompletely polarized (B) MDCK and ConA^r cells were determined and normalized to that for AP-side-infected, untreated MDCK cells. Percentages of LDH release in well-polarized (C) and incompletely polarized (D) Calu-3 cells were determined and normalized to AP-side-infected, untreated Calu-3 cells. *, P < 0.05 compared to AP-side-infected, untreated MDCK cells; ×, P < 0.05 compared to AP-side-infected, untreated ConA^r cells; +, P < 0.05 compared to AP-side-infected, untreated Calu-3 cells; **, P < 0.05 compared to BL-side-infected, untreated MDCK cells; ××, P < 0.05 compared to BL-side-infected, untreated ConA^r cells; ++, P < 0.05 compared to BL-side-infected, untreated Calu-3 cells.

P. aeruginosa internalization is dependent on bacterial binding to N-glycans on the AP surface of well-polarized cells, to HSPGs on the BL surface of well-polarized cells, and to both molecules at the AP surface of incompletely polarized cells. Cells were pretreated with the indicated sugars to upregulate N-glycosylation (A and B), with the indicated concentrations of tunicamycin to inhibit N-glycosylation (C and D), or with the indicated amounts of heparinase III to remove HS chains (E to H). Bacteria were added for 1 h, and standard invasion assays were performed. Shown are the means ± SD for three separate experiments. (A and B) Man and Glc are sufficient to enhance bacterial invasion of MDCK, ConA^r, and Calu-3 cells from the AP surface. (A) P. aeruginosa internalization in well-polarized MDCK and ConA^r cells, normalized to AP-side-infected, untreated MDCK cells. (B) P. aeruginosa internalization in well-polarized Calu-3 cells, normalized to AP-side-infected, untreated cells. (C and D) Tunicamycin inhibits invasion from the AP surface of well-polarized MDCK and Calu-3 cells in a dose-dependent manner. (C) P. aeruginosa internalization in well-polarized MDCK and ConA^r cells, normalized to AP-side-infected, untreated MDCK cells. (D) P. aeruginosa internalization in well-polarized Calu-3 cells, normalized to AP-side-infected, untreated cells. (E to H) Heparinase III treatment inhibits invasion from the BL surface of well-polarized and incompletely polarized MDCK, ConA^r, and Calu-3 cells and from the AP surface of incompletely polarized cells. P. aeruginosa binding to well-polarized (E) and incompletely polarized (F) MDCK and ConA^r cells was determined and normalized to that of AP-side-infected, untreated MDCK cells. P. aeruginosa binding to well-polarized (G) and incompletely polarized (H) Calu-3 cells was determined and normalized to that of AP-side-infected, untreated cells. *, P < 0.05 compared to AP-side-infected, untreated MDCK cells; ×, P < 0.05 compared to AP-side-infected, untreated ConA^r cells; +, P < 0.05 compared to AP-side-infected, untreated Calu-3 cells; **, P < 0.05 compared to BL-side-infected, untreated MDCK cells; ××, P < 0.05 compared to BL-side-infected, untreated ConA^r cells; ++, P < 0.05 compared to BL-side-infected, untreated Calu-3 cells.

P. aeruginosa colocalizes with HS-rich patches on the exposed BL membrane and with N-glycan-rich patches on the exposed AP membrane of highly polarized 3D cysts. (A and B) Representative x-y confocal sections of BL-side-out or AP-side-out MDCK cysts cultured for 5 days and stained with anti-HS antibody (HS; purple) or ConA (Man; purple) and with phalloidin (actin; red). (A) BL-side-out (left) and AP-side-out (right) MDCK cysts show preferential localization of HSPGs to the BL surface of the cysts. (B) BL-side-out (left) and AP-side-out (right) MDCK cysts show localization of Man to both the AP and BL surfaces of the cysts. (C to F) 3D reconstructions of z-stack images acquired by confocal microscopy. HS is stained with an anti-HS antibody (HS; purple), Man is stained with ConA (Man; purple), and actin is stained with phalloidin (actin; red). (C) BL-side-out MDCK cysts show uniform staining of HS at the exposed BL membrane. (D) MDCK cysts treated with heparinase III and infected with GFP-PAK cells for 2 h (green) show nonuniform patchy HS staining, and the bacteria bind preferentially to HS-rich areas (purple). (E) BL-side-out wt MDCK cysts show uniform staining of Man at the exposed BL membrane. (F) BL-side-out ConA^r MDCK cysts briefly treated with Man (to induce expression of more complex N-glycans) and infected with GFP-PAK cells for 2 h (green) show nonuniform patchy Man staining, and the bacteria do not bind preferentially to Man-rich areas (purple). (G) Quantification of GFP-PAK bound to HS-rich versus HS-poor patches in BL-side-out MDCK cysts pretreated with heparinase III. (H) Quantification of bacteria bound to Man-rich versus Man-poor patches in BL-side-out ConA^r cysts briefly treated with Man. (I and J) 3D reconstructions of z-stack images of ConA^r MDCK cysts acquired by confocal microscopy. Man is stained with ConA (purple), and actin is stained with phalloidin (red). (I) AP-side-out ConA^r cysts infected with PAK-GFP cells show nonuniform patchy staining of Man at the exposed AP membrane, and the bacteria do not bind preferentially to Man-rich areas. (J) ConA^r cysts briefly treated with Man and infected with GFP-PAK cells (green) show nonuniform patchy Man staining, and the bacteria bind preferentially to Man-rich areas (purple). (K) Quantification of GFP-PAK bound to Man-rich versus Man-poor patches in AP-side-out ConA^r cysts. (L) Quantification of GFP-PAK bound to Man-rich versus Man-poor patches in AP-side-out ConA^r cysts briefly treated with Man. *, P < 0.01.