Tfp-deficient P. aeruginosa mutant binds preferentially to HS at the BL surface while flagella-deficient mutant binds preferentially to N-glycans at the AP surface of polarized epithelium

Our previous studies established that P. aeruginosa binds preferentially to N-glycan chains at the AP surface of polarized epithelium, with preferential affinity for more complex chains after up-regulations of N-glycosylation [9]. At the BL surface, the bacterium binds preferably to HS chains of HSPGs. To determine whether flagella or Tfp, the major P. aeruginosa adhesins, differentially mediate binding to these distinct AP and BL host receptors, we utilized isogenic mutants of PAO1 in which the gene encoding PilA, the major subunit of the Tfp (PAO1ΔpilA) or the gene encoding FliC, the major subunit of flagella (PAO1ΔfliC), is deleted. Standard adhesion assays, in which bacteria were added for 2 h to the AP or BL surface of polarized Calu-3 cells grown as polarized monolayers on Transwell filters, were performed [9]. While other P. aeruginosa adhesins have been identified, such as the cup fimbrial adhesins [35] and lectins PA-IL (LecA) and PA-IIL (LecB) [36], Tfp and flagella were the predominant adhesins under the conditions of our experiments, as the PAO1ΔfliCΔpilA double mutant exhibited undetectable binding (data not shown and [21]).

Consistent with our previously published studies utilizing PAK [9], PAO1 bound approximately 2-fold more efficiently to the BL surface than to the AP surface of polarized airway epithelium (Figure 1A). Both the Tfp mutant (PAO1ΔpilA) and the flagella mutant (PAO1ΔfliC) bound less efficiently to the AP or BL surfaces of the epithelium when compared to PAO1 (Figures 1A–C), suggesting that the absence of either of these two adhesins impacts bacterial binding and that Tfp and flagella may function synergistically. Importantly, PAO1ΔfliC still bound almost 2-fold better than PAO1ΔpilA to the AP surface (P<0.05), but it bound ∼9-fold less efficiently than PAO1ΔpilA to the BL surface (P<0.05) (compare Figures 1B and C). Furthermore, while PAO1ΔpilA bound ∼3-fold more efficiently to the BL surface than to the AP surface of polarized epithelium (Figure 1B), PAO1ΔfliC adhered over 5-fold better to the AP surface than to the BL surface (Figure 1C). These results suggest that Tfp are the predominant adhesin at the AP surface whereas flagella function as the major adhesin at the BL surface.

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Figure 1. PAO1ΔpilA binds preferentially to HS at the BL surface and PAO1ΔfliC binds preferentially to N-glycans at the AP surface.

Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparin, heparinase III (hepIII), mannose (Man), or tunicamycin (tun). Host cell treatments are color-coded: treatments that affect HSPGs are indicated with shades of red and treatments that affect N-glycans are indicated with shades of blue. (A) PAO1, (B) PAO1ΔpilA, or (C) PAO1ΔfliC were added to the AP or BL chamber for 2 h and standard adhesion assays were performed. Shown is the mean +/− SD for 4 independent experiments. ^*P<0.05 compared to cells infected with PAO1 at the AP surface (black bar). ^**P<0.05 compared to cells infected with PAO1 at the BL surface (black bar).

https://doi.org/10.1371/journal.ppat.1002616.g001

We then investigated the role of host N-glycans in Tfp- or flagella-mediated binding. N-glycans are found on both AP and BL surfaces of polarized epithelium whereas HS chains are expressed predominantly on the BL surface of polarized epithelium [9]. When the expression of more complex N-glycans was triggered by long-term culture of Calu-3 cells in the presence of mannose (Man) [37], we observed a 2-fold increase in the binding of PAO1 or PAO1ΔfliC to the AP surface of polarized airway epithelium. However, no effect on the BL binding of PAO1 or PAO1ΔfliC was observed (Figure 1C). In control experiments, long-term culture of Calu-3 cells in the presence of galactose (Gal), which does not enhance N-glycosylation, had no effect on bacterial adhesion (data not shown). Inhibition of N-glycosylation by pre-treatment of Calu-3 cells with tunicamycin, which decreased N-glycosylation by 50% under the conditions of our experiments (more extensive deglycosylation disrupted the monolayer integrity), decreased the AP adhesion of PAO1 (Figure 1A) and PAO1ΔfliC (Figure 1C) by 2.5-fold, but had no effect on BL binding. These treatments did not have statistically significant effects on binding of PAO1ΔpilA to the AP surface of polarized cells (Figure 1B). Together, these results suggest that Tfp bind primarily to N-glycans at the AP surface of polarized epithelial cells.

We used two approaches to determine the role of host HS chains of HSPGs in Tfp- and flagella-mediated bacterial adherence. First, addition of excess heparin competitively inhibited the binding of PAO1 and PAO1ΔpilA at the BL surface of Calu-3 cells (Figures 1A and B), but had no effect on binding of PAO1ΔfliC to the BL surface (Figure 1C). Since HSPGs are predominantly expressed on the BL surface, it was not surprising to observe that exogenous addition of heparin had little effect on binding of PAO1 or of either adhesin mutant to the AP surface of polarized cells. To rule out non-specific charge effects, we demonstrated that addition of another highly negatively charged glycosaminoglycan chain, chondroitin sulfate (CS), had no effect on binding of PAO1, PAO1ΔpilA, or PAO1ΔfliC to either surface (data not shown). Second, pre-treatment of cells with heparinase III, an enzyme that cleaves HS chains, had a similar effect on bacterial adhesion as did addition of excess heparin. It reduced adhesion of PAO1 and PAO1ΔpilA to the BL surface of polarized Calu-3 cells but had no effect on adhesion of PAO1ΔfliC. Heparinase III treatment had no significant effect on the binding of any of the strains to the AP surface of polarized Calu-3 cells. Enzymatic removal of CS by chondroitinase ABC did not alter bacterial attachment at either surface (data not shown), confirming the specific role of HS in flagella-mediated binding of P. aeruginosa. Together, these results demonstrate P. aeruginosa binding at the BL surface is predominantly mediated by flagella interactions with HS chains of HSPGs.

Flagella-coated beads bind directly to HS while Tfp-coated beads bind directly to N-glycans in vitro

Two different in vitro binding affinity assays were utilized to rigorously determine whether Tfp or flagella were sufficient to mediate binding to N-glycans or HS, respectively. We have previously developed a fluorometric assay that quantifies bacterial attachment to plastic wells coated with various glycans [9]. Our studies revealed that GFP-expressing PAO1 binds in a dose-dependent manner to plastic wells coated with HS or to a complex hybrid N-glycan chain ((Gal-GlcN)₄Man₃(GlcN)₂), with the strongest binding observed to HS. Here, we modified this assay; in place of bacteria, we used 2-µm green fluorescent beads coated with purified flagella or Tfp, isolated by shearing from PAO1ΔpilA or PAO1ΔfliC, respectively (Figure S1). Coommasie Blue staining of the purified adhesin preparations electrophoresed on SDS-PAGE did not reveal any contaminating products (Figure S1). We note that the sheared adhesins may be composed of short polymers (i.e. flagella or Tfp) or the individual subunits (flagellin or pilin). For ease of clarity, we will refer to them as flagella or Tfp.

As shown in Figure 2B, flagella-coated beads bound strongly in a dose-dependent manner to HSPG or HS chains alone and at low levels to different N-glycans (structures shown in Figure 2A), but not measurably to single sugars (Man, N-acetylated glucosamine (GlcNAc), fucose Fuc, or galactose (Gal)), CS, or to non-sulfated hyaluronic acid (HA). These results suggest that i) flagella are capable of strong binding to HS chains, that ii) sulfate groups of HS provide binding sites and/or the anionic charge of sulfate is necessary for the interaction, and that iii) flagella may also bind to sugar sequences along the N-glycan chain in a polyvalent manner.

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Figure 2. Flagella-coated beads bind directly to HS and Tfp-coated beads bind directly to N-glycans in vitro.

(A) The structure of different N-glycan chains used in the study (adapted from the manufacturer). (B) Flagella- or (C) Tfp-coated beads were added to 96-well plastic plates coated with increasing concentrations of various molecules for 1 h. The fluorescence of the bound fraction was quantified in a plate reader and the percent of binding above control (binding of coated beads to non-coated wells) is indicated. Shown is the mean +/− SD for 6 independent experiments. (D) Flagella- or (E) Tfp-coated green beads were mixed with red beads coated with the indicated molecules, mixed gently for 2 h, and examined by IF. Exogenous heparin was added to competitively block flagella-HS aggregation, and exogenous N-glycan-3 to block flagella-N-glycan-3 and Tfp-N-glycan-3 aggregation. Yellow clumps indicate aggregation of green and red beads. The fraction of green aggregates, red aggregates, and mixed (yellow) aggregates from 3 separate experiments is shown beneath each panel. HSPGs: heparan sulfate proteoglycans, HS: heparan sulfate; HA: hyaluronic acid; CS-4: 4-0-sulfated chondroitin sulfate; CS-6: 6-0-sulfated chondroitin sulfate; N-glycan-1: simple N-glycan chain; N-glycan-2: hybrid N-glycan chain; N-glycan-3: complex N-glycan chain; Man: mannose; GlcNAc: N-acetylglucosamine; Fuc: fucose; Gal: galactose; BSA: bovine serum albumin.

https://doi.org/10.1371/journal.ppat.1002616.g002

We next examined the binding specificity of beads coated with sheared Tfp. In contrast to the results obtained with flagella-coated beads, Tfp-coated beads bound most avidly to N-glycan chains (Figure 2C), with the strongest binding to N-glycan-3, the most complex N-glycan chain (Figure 2A). There was minimal binding to the single sugars (Man, GlcNAc, Fuc, or Gal) indicating that single sugars are not sufficient to mediate binding to Tfp. Likewise, there was almost no binding to HSPGs, HS, or other glycosaminoglycans, suggesting that Tfp almost exclusively recognize N-glycan chains. Identical results were obtained with beads coated with flagella or Tfp isolated from strain PAK (Figure S2) and with pili isolated from strain PA103 (Figure S3), suggesting that the observed binding specificities are a general property of the adhesins and are not strain-specific.

As a further test for the specificity of the binding of Tfp-coated beads, we tested whether the C-terminus of Tfp, which contains the binding determinants, was required. For these experiments, we isolated sheared surface Tfp from a PA103 mutant, which harbors a transposon insertion 13 amino acids from the C-terminus (PA103 Mutant 9 [38]) (Figure S3). The mutant pilin is predicted to be truncated between the two C-terminal cysteines required for pilin folding [39]. Beads coated with Tfp isolated from PA103 pili (Figure S3) demonstrated a similar binding specificity as beads coated with Tfp from PAO1 (Figure 2) or PAK (Figure S2). In contrast, the Mutant 9 Tfp-coated beads bound poorly to N-glycans (Figure S3). These results suggest that three-dimensional structure of pili is not compromised under the conditions of our experiments and that the C-terminal binding determinants are required for in vitro binding of Tfp to N-glycans.

We extended our studies using a complementary in vitro assay that measures bead aggregation under defined shear forces to detect molecular interactions [40]. As shown in Figure 2D, we first tested the interaction of flagella-coated green fluorescent beads with red fluorescent beads coated with various glycans. In control experiments in which equal numbers of flagella-coated green beads were mixed with BSA-coated red beads, 99% of the aggregates comprised a single color (52% green and 47% red) and only 1% were yellow. These results indicate that there is minimal non-specific bead aggregation under the conditions of our experiments. In contrast, when flagella-coated green beads were mixed with HSPG-coated red beads, 80% of the aggregates were yellow. This interaction was inhibited by exogenous addition of heparin, with the number of yellow aggregates decreasing to ∼10%. Aggregation of flagella-coated green beads with N-glycan-3-coated red beads resulted in 34% yellow aggregates, which was decreased to 16% when excess N-glycan-3 was added to competitively inhibit binding. Together, both of these in vitro assays confirm our cell-culture based experiments and demonstrate conclusively that flagella can bind directly to HS and, to much lesser extent, to complex N-glycan chains.

We next examined the binding of Tfp-coated green beads to glycan-coated red beads (Figure 2E). Whereas Tfp-coated green beads mixed with BSA-coated red beads resulted in only 2% mixed yellow aggregates, incubation with N-glycan-3-coated red beads resulted in 74% yellow aggregates, which was decreased to 12% upon addition of excess N-glycan-3 (Figure 2E). We also tested the ability of Tfp-coated green beads to interact with red beads coated with individual sugars. Mixing Tfp-coated green beads with Man-coated red beads resulted in only 25% yellow aggregates, and in 16% yellow aggregates when Tfp-coated green beads were mixed with Gal-coated red beads. Importantly, Tfp-coated green beads did not aggregate with HS-coated red beads. These findings confirm that Tfp preferentially interact directly with complex N-glycans in a polyvalent manner and individual sugars do not provide enough strength and specificity for the interaction. Taken together, in vitro binding assays conclusively show that Tfp are necessary and sufficient to interact with N-glycans, which corroborates our cell-culture based experiments.

Flagella or Tfp are sufficient to mediate binding to HS- or N-glycan-rich areas, respectively, in polarized epithelium

Our results thus far suggest that Tfp are necessary in vivo and sufficient in vitro to interact with N-glycans whereas flagella are necessary in vivo and sufficient in vitro to interact with HS. As it is possible that additional host molecules mediate bacterial binding to cultured cells, we tested by IF microscopy whether Tfp-coated or flagella-coated beads would be sufficient to mediate binding to the AP surface of airway epithelial cells and compared these results with the binding of PAO1ΔpilA and PAO1ΔfliC. For these experiments we utilized Calu-3 cells grown as confluent monolayers on Transwell filters for a shorter time period (3 days rather than the usual 9 days). Under these conditions, functional tight junctions are formed, but the polarized distribution of HSPGs is not complete, i.e. some HSPGs are still present at the AP surface [9]. These 3-day grown monolayers, which we term incompletely polarized, were briefly treated with heparinase III or tunicamycin to further decrease the surface presentation of HSPGs or N-glycans, respectively. The resulting patchy distribution of HSPGs or N-glycans at the AP surface allowed us to correlate and quantify by IF the binding of PAO1 flagella or Tfp mutants, or adhesin-coated beads, to HS-rich or -poor areas (visualized with an antibody to HS) or N-glycan-rich or -poor areas (visualized with fluorescent lectin that binds to Man residues in N-glycan chains).

We first examined the binding of GFP-expressing PAO1ΔpilA and PAO1ΔfliC to membrane regions rich in HS or N-glycan chains. As shown in Figures 3A and B, significantly more PAO1ΔpilA-GFP co-localized to HS-rich patches (65%) than to HS-poor patches (35%) on the AP surface of heparinase III-treated Calu-3 monolayers. In contrast, only ∼30% of PAO1ΔfliC-GFP bound to HS-rich patches whereas ∼70% co-localized with HS-poor patches. In Calu-3 cells briefly treated with tunicamycin, only ∼40% of PAO1ΔpilAI-GFP co-localized to N-glycan-rich patches whereas a much larger fraction, almost 70%, of PAO1ΔfliC-GFP co-localized to N-glycan-rich areas (Figures 3C and D). In summary, PAO1ΔpilA, for which flagella serve as the major adhesin, preferentially co-localized with HS-rich areas. In contrast, PAO1ΔfliC, for which Tfp serve as the major adhesin, preferentially co-localized with N-glycan-rich areas at the AP surface of incompletely polarized epithelium.

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Figure 3. Flagella mediate co-localization with HS-rich regions while Tfp mediate co-localization with N-glycan-rich areas in polarized epithelium.

Calu-3 cells were grown as incompletely polarized monolayers on Transwell filters for 3 days and briefly treated with heparinase III or with tunicamycin. Binding of bacteria or adhesin-coated beads to the AP surface was examined by confocal microscopy with 3D reconstructions of Z stack images and quantification of bacterial or coated-bead localization to HS-rich, HS-poor, N-glycan-rich, or N-glycan-poor regions of the AP surface was determined. HS was visualized with an anti-HS antibody (purple), N-glycans with concanavalin A (purple), actin with phalloidin (red). (A) Co-localization and (B) quantification of PAO1ΔpilA-GFP or PAO1ΔfliC-GFP binding to HS-rich (purple) or HS-poor (red) areas. Black circles show co-localization of P. aeruginosa mutants with HS-rich areas. (C) Co-localization and (D) quantification of PAO1ΔpilA-GFP or PAO1ΔfliC-GFP binding to N-glycan-rich (purple) or N-glycan-poor (red) areas. Black circles show co-localization of P. aeruginosa mutants with N-glycan-rich areas. (E) Co-localization and (F) quantification of flagella- or Tfp-coated green beads binding to HS-rich or HS-poor areas. Black circles show co-localization of flagella- or Tfp-coated green beads with HS-rich areas. (G) Co-localization and (H) quantification of flagella- or Tfp-coated green beads binding to N-glycan-rich (purple) or N-glycan-poor (red) areas. Black circles show co-localization of flagella- or Tfp-coated green beads with N-glycan-rich areas. Shown is the mean +/− SD for 3 independent experiments. ^*P<0.01.

https://doi.org/10.1371/journal.ppat.1002616.g003

We then tested whether Tfp or flagella were sufficient to mediate these specific interactions by quantifying the binding of Tfp- or flagella- coated green fluorescent beads to N-glycan or HSPG-rich areas at the AP surface. As shown in Figures 3E and F, 75% of flagella-coated beads co-localized with HS-rich areas at the AP surface of heparinase III-treated Calu-3 cells. In stark contrast, only 15% of Tfp-coated beads co-localized with HS-rich patches. The opposite results were observed for binding to N-glycans: ∼30% of flagella-coated beads compared to 80% of Tfp-coated beads co-localized with N-glycan-rich areas (Figures 3G and H). Importantly, the results paralleled what was observed with intact bacteria. Altogether, these experiments demonstrate that Tfp are sufficient to mediate P. aeruginosa binding to N-glycan chains and that flagella are sufficient to mediate P. aeruginosa binding to HS chains of HSPGs at the surface of airway epithelium.

Bacterial internalization is mediated by Tfp-dependent binding to N-glycans at the AP surface or by flagella-dependent binding to HS at the BL surface of polarized epithelium

Following binding to the epithelium, P. aeruginosa is able to enter into non-phagocytic cells; this event is most readily detectable in ∼75% of clinical, environmental, and laboratory strains, including PAO1 or PAK, that do not secrete the potent phospholipase ExoU but that encode ExoS [31], [41]. In order to examine the role of flagella and Tfp interactions during entry at the AP or BL surface, polarized Calu-3 cells were pre-treated with various agents and standard bacterial invasion assays were performed. In general, the results of the invasion assays with PAO1, PAO1ΔpilA, and PAO1ΔfliC were directly proportional to adhesion assays (see Figure 1), i.e., more binding resulted in more invasion. Competitive inhibition with heparin or enzymatic cleavage of HS by heparinase III reduced PAO1 and PAO1ΔpilA invasion at the BL, but not AP surface, of polarized cells (Figures 4A and B). Up-regulation of N-glycosylation by long-term culture of Calu-3 cells in the presence of Man enhanced the internalization of PAO1 or PAO1ΔfliC at the AP surface of polarized cells, while inhibition of N-glycosylation with tunicamycin reduced bacterial entry at the AP surface of polarized Calu-3 cells (Figures 4A and C). Notably, inhibition of N-glycosylation had a small but statistically significant effect on PAO1 and PAO1ΔpilA entry at the BL surface, although it did not have any effect on bacterial binding. This finding suggests that flagella-dependent entry, but not binding, at the BL surface may be mediated by a yet unidentified N-glycosylated receptor(s). Together, these data confirm that P. aeruginosa-induced binding and subsequent entry into polarized epithelium are primarily mediated by Tfp-dependent binding to host N-glycans at the AP surface and by flagella-dependent binding to HS chains at the BL surface of polarized epithelium.

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Figure 4. P. aeruginosa internalization at the AP surface of polarized epithelium is mediated by Tfp-dependent binding to N-glycans and subsequent PI3K activation and, at the BL surface, by flagella-dependent binding to HS and subsequent EGFR/PI3K activation.

Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparin, heparinase III (hepIII), mannose (Man), tunicamycin (tun), EGFR inhibitor (AG1478), PI3K inhibitor (LY29004), or in combination. (A) PAO1, (B) PAO1ΔpilA or (C) PAO1ΔfliC were added to the AP or BL chamber for 2 h and standard invasion assays were performed. Shown is the mean +/− SD for 4 independent experiments. ^*P<0.05 compared to cells infected with PAO1 at the AP surface (black bar). ^**P<0.05 compared to cells infected with PAO1 at the BL surface (black bar).

https://doi.org/10.1371/journal.ppat.1002616.g004

Flagella-mediated bacterial entry at the BL surface and Tfp-mediated entry at the AP surface of polarized epithelium involve PI3K/Akt activation

We have previously shown that P. aeruginosa internalization at the AP surface of incompletely polarized MDCK cells is actin-dependent and requires activation of PI3K and its effector protein Akt [33]. We therefore determined whether N-glycans and/or HSPGs acted as host receptors upstream of this signaling pathway and whether bacterial Tfp and flagella were bacterial partners associated with the PI3K/Akt-dependent entry at the AP or BL surface of polarized lung airway epithelial cells. Pre-treatment with LY29004, an inhibitor of PI3K, did not affect PAO1 binding at either the AP or BL surface of fully polarized Calu-3 cells (Figure S4). However, it had a pronounced effect on bacterial invasion at the BL surface, reducing it ∼5 fold, and it had a smaller but statistically significant effect on invasion at the AP surface (Figure 4A.). PI3K-dependent entry at the BL surface required flagella binding to HS, as inhibition of PI3K, competitive inhibition with heparin, or heparinase-III treatment decreased PAO1ΔpilA entry similarly to PAO1, but had no effect on the already low levels of PAO1ΔfliC entry (Figures 4B and C). At the AP surface, inhibition of PI3K caused a small but statistically significant decrease in PAO1ΔfliC invasion. Simultaneous PI3K inhibition and tunicamycin treatment did not further reduce PAO1ΔfliC entry (Figure 4C), suggesting that PI3K-dependent invasion at the AP surface could require Tfp-mediated binding to N-glycans. Together, these results suggest that flagella-mediated binding at the BL surface leads to P. aeruginosa internalization through a PI3K-dependent pathway. At the AP surface, Tfp mediated entry through a PI3K-dependent entry can also occur, although consistent with our previously published results [9], BL entry is more efficient than AP entry.

On the basis of our results, we would predict that flagella-mediated binding and entry at the BL surface or Tfp-mediated binding and entry at the AP surface should increase downstream Akt phosphorylation. To test this hypothesis, polarized Calu-3 cells were co-cultivated for 60 min with PAO1 or with adhesin mutants, and Akt was immunoprecipitated followed by immunoblotting with anti-phospho Akt^Ser473 antibody. The ratio of phospho-Akt to total Akt was quantified and normalized to the ratio observed in untreated cells. In control experiments, Calu-3 cells were AP or BL exposed to heparin-binding EGF-like growth factor (HB-EGF) for 10 min since Akt phosphorylation is well established as a downstream consequence of EGFR activation. BL addition of HB-EGF increased Akt phosphorylation over 2-fold, but had little effect when added to the AP surface (Figures 5A and B), which is consistent with the known BL localization of EGFR in polarized epithelium and, thus, BL activation of Akt. Addition of PAO1 or PAO1ΔpilA at the BL surface resulted in activation of Akt, with a 2.5-fold increase in the fraction of phosphorylated Akt when compared to bacterial addition to the AP surface (Figures 5A–D). However, AP addition of PAO1ΔpilA or BL addition of PAO1ΔfliC failed to activate Akt (Figures 5C–F). This observation is consistent with a requirement for initial Tfp-mediated binding at the AP surface and flagella-mediated binding at the BL surface. Inhibition of PI3K prior to the addition of bacteria almost completely eliminated Akt phosphorylation at the AP and BL surface (Figures 5A–F), consistent with the known role of PI3K and Akt activation in P. aeruginosa entry [33].

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Figure 5. Akt is phosphorylated upon flagella-mediated P. aeruginosa entry at the BL surface and upon Tfp-mediated entry at the AP surface of polarized epithelium.

Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparinase III (hepIII), tunicamycin (tun), EGFR inhibitor (AG1478), or PI3K inhibitor (LY29004). As a control, cells were left untreated (un) or, as a positive control, cells were treated with HB-EGF. (A, B) PAO1, (C, D) PAO1ΔpilA, or (E, F) PAO1ΔfliC were added to the AP or BL chamber for 1 h. Lysates were immunoprecipitated with Akt antibody followed by immunoblotting with phospho- or total-Akt antibodies. Representative gels (A, C, E) and quantification by densitometry of three gels (B, D, F) are shown. The ratio of phospho-Akt to total-Akt for untreated cells was set to 1. Shown is the mean +/− SD for 3 independent experiments. ⁺P<0.05 compared to cells AP infected with PAO1. ⁺⁺P<0.05 compared to cells BL infected with PAO1. ^*P<0.05 compared to cells AP infected with PAO1 (B), PAO1ΔpilA (D), or PAO1ΔfliC (F). ^**P<0.05 compared to cells BL infected with PAO1 (B), PAO1ΔpilA (D), or PAO1ΔfliC (F).

https://doi.org/10.1371/journal.ppat.1002616.g005

We next determined whether Tfp or flagella-mediated activation of Akt involved HSPGs or N-glycans. Pre-treatment of Calu-3 cells with heparinase III reduced Akt phosphorylation to near basal levels upon BL, but not AP, addition of PAO1 or PAO1ΔpilA (Figures 5A–D). Inhibition of N-glycosylation with tunicamycin partially reduced Akt activation upon AP addition of PAO1 and PAO1ΔfliC (Figures 5A–B and E–F). Interestingly, inhibition of N-glycosylation reduced Akt phosphorylation upon BL infection with PAO1 or PAO1ΔpilA, which is consistent with our bacterial invasion results (see Figure 4) and suggests involvement of yet unidentified N-glycosylated receptor in flagella-HS-dependent invasion and activation of PI3K/Akt pathway. Together, these results strongly suggest that activation of PI3K/Akt pathway at the BL surface is primarily dependent on flagella-mediated bacterial binding to HS chains of HSPGs, while at the AP surface it is dependent on Tfp-mediated bacterial binding to N-glycan chains.

Flagella-mediated P. aeruginosa entry at the BL surface involves EGFR activation

While many stimuli can activate the PI3K/Akt pathway, we were particularly interested in interrogating whether flagella- or Tfp-mediated binding to HSPGs or N-glycan chains and/or subsequent bacterial entry involved growth factor receptors (GFRs). Notably, GFRs require N-glycosylation for their activity and many of them are also modulated by HSPGs; our work clearly establishes the role of both N-glycans and HSPGs for P. aeruginosa binding and internalization.

In preliminary studies, we investigated the role of epidermal GFR (EGFR), platelet-derived GFR (PDGFR), and fibroblast GFR (FGFR). While pharmacologic inhibition of any of the GFRs did not affect bacterial binding, inhibition of EGFR and PDGFR, but not FGFR, reduced bacterial internalization at the BL surface of Calu-3 cells (Figure S4). We also confirmed the role of EGFR and PDGFR in P. aeruginosa entry using siRNA gene silencing in HeLa cells (Figure S4). Simultaneous removal of HS by heparinase III and pharmacologic inhibition of EGFR had the same effect as heparinase III treatment alone (Figure S4). These results suggest that EGFR may potentially mediate bacterial entry upon HS-dependent bacterial binding, and thus EGFR was a logical candidate to study further.

To elucidate the role of EGFR in flagella- and Tfp-mediated bacterial internalization, we first showed that inhibition of EGFR reduced invasion of PAO1 and PAO1ΔpilA at the BL surface of polarized Calu-3 cells, but did not further decrease the invasion of PAO1ΔfliC (Figure 4). Inhibition of EGFR did not have any effect on bacterial internalization at the AP surface since EGFR is predominantly expressed on the BL surface of polarized epithelium. Simultaneous inhibition of N-glycosylation and pharmacologic inhibition of EGFR reduced PAO1 and PAO1ΔpilA internalization at the BL surface similarly to what was observed with each treatment alone (Figures 4A and B), confirming that EGFR activity depends on its N-glycosylation. Inhibition of PAO1ΔpilA entry by heparin, heparinase III treatment, or PI3K inhibition reduced bacterial entry to similar degrees, while EGFR inhibition had somewhat intermediate effect (Figure 4B). Furthermore, combined heparinase III treatment and EGFR inhibition reduced PAO1 and PAO1ΔpilA internalization to a greater degree than inhibition of EGFR alone (Figures 4A and B). These results suggest that bacterial internalization occurs through multiple HSPG-dependent pathways, including one that involves a flagella-HS-EGFR complex leading to PI3K activation at the BL surface of polarized epithelium.

To further investigate the role of flagella or Tfp during EGFR-dependent entry at the AP or BL surface of polarized epithelial cells, we tested whether bacterial binding induced EGFR phosphorylation. Total EGFR was immunoprecipitated from cell lysates 1 h after AP or BL infection with PAO1, PAO1ΔpilA or PAO1ΔfliC, followed by immunoblotting with a monoclonal anti-phospho EGFR^Ser1046/1047 antibody. The ratio of phospho-EGFR to total EGFR was quantified and normalized to the ratio observed in untreated cells. In control experiments, BL exposure of Calu-3 cells to HB-EGF for 10 min increased EGFR phosphorylation 3-fold, but had little effect when applied to the AP surface (Figures 6A and B), consistent with the known BL localization of EGFR in polarized epithelium. Binding of PAO1 or PAO1ΔpilA to the BL surface of polarized epithelium resulted in a 2- to 2.5-fold increase in the fraction of phospho-EGFR (Figures 6A–D). In contrast, binding of PAO1ΔfliC failed to increase EGFR phosphorylation above background levels (Figures 6E and F), suggesting that flagella-mediated binding is required for EGFR activation. Activation of EGFR upon BL addition of PAO1 or PAO1ΔpilA was reduced by EGFR inhibition, removal of HS by heparinase III, or inhibition of N-glycosylation by tunicamycin, confirming the involvement of bacterial flagella, host HSPGs, and N-glycosylation in bacteria-mediated EGFR phosphorylation.

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Figure 6. EGFR is phosphorylated upon flagella-mediated P. aeruginosa entry at the BL surface of polarized epithelium.

Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparinase III (hepIII), tunicamycin (tun), or EGFR inhibitor (AG1478). As a control, cells were left untreated (un) or, as a positive control, cells were treated with HB-EGF. (A, B) PAO1, (C, D) PAO1ΔpilA, or (E, F) PAO1ΔfliC were added to the AP or BL chamber for 1 h. Lysates were immunoprecipitated with EGFR antibody followed by immunoblotting with phospho- or total-EGFR antibodies. Representative gels (A, C, E) and quantification by densitometry of three gels (B, D, F) are shown. The ratio of phospho-EGFR to total-EGFR for untreated cells was set to 1. Shown is the mean +/− SD for 3 independent experiments. ^**P<0.05 compared to cells BL infected with PAO1 (B) or PAO1ΔpilA (D).

https://doi.org/10.1371/journal.ppat.1002616.g006

Finally, we tested whether flagella-mediated PI3K activation was EGFR dependent. Indeed, inhibition of EGFR decreased Akt phosphorylation in response to BL addition of PAO1 or PAO1ΔpilA, but not PAO1ΔfliC (Figures 5A–D). These results indicate that P. aeruginosa-mediated internalization at the BL surface occurs principally via flagella-mediated interactions through a pathway that utilizes HSPGs and that involves EGFR and PI3K/Akt activation. At the AP surface, Tfp mediates bacterial entry through a PI3K/Akt pathway that is independent of EGFR.

Flagella are sufficient to induce EGFR phosphorylation

To elucidate whether Tfp and/or flagella alone were sufficient to induce phosphorylation of EGFR, beads coated with purified adhesins were added to polarized Calu-3 cells for 1 h and immunoprecipitation/immunoblotting assays were performed as previously for the whole bacteria. Remarkably, addition of flagella-coated beads to Calu-3 cells increased the fraction of phospho-EGFR (1.7-fold) (Figures 7 C and D), almost to levels seen upon BL addition of PAO1 (2.2-fold) (Figures 6 A and B). In contrast, no increase in the ratio of phospho-EGFR was observed upon addition of the flagella-coated beads to the AP surface or upon AP or BL addition of BSA-coated beads. Similar to what we observed with PAO1 or PAO1ΔpilA, flagella-mediated phosphorylation of EGFR was reduced after inhibition of EGFR, HS removal by heparinase III, or inhibition of N-glycosylation by tunicamycin (Figures 7C and D). Notably, AP or BL addition of Tfp-coated beads did not detectably increase phospho-EGFR above background levels (Figures 7E and F). We were unable to detect induction of Akt phosphorylation by either flagella- or Tfp-coated beads (Figures 7A and B). Altogether, these results demonstrate that flagella alone are sufficient to induce EGFR phosphorylation.

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Figure 7. Flagella are sufficient to phosphorylate EGFR and Shc.

Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparinase III (hepIII), tunicamycin (tun), or EGFR inhibitor (AG1478). As a control, cell were left untreated (un); flagella-, Tfp-, or, as a negative control, BSA-coated beads were added to the AP or BL chamber for 1 h. Lysates were immunoprecipitated with Akt or EGFR antibody or directly immunoblotted with (A, B) phospho-Akt, (C–F) phospho-EGFR, or (G–H) phospho-Shc (three different isoforms p46, p52, and p66). Representative gels (A, C, E, G) and quantification by densitometry of three gels (B, D, F, H) are shown. The ratio of phospho-Akt to total-Akt, phospho-EGFR to total-EGFR, or phospho-Shc to total-Shc for untreated cells was set to 1. Shown is the mean +/− SD for 3 independent experiments. ^**P<0.05 compared to cells BL infected with PAO1.

https://doi.org/10.1371/journal.ppat.1002616.g007

To confirm the specificity and significance of EGFR phosphorylation triggered by flagella-coated beads, we tested other targets of EGFR activation. As shown in Figures 7G and H, addition of flagella-coated beads to the BL surface of polarized epithelium resulted in phosphorylation of EGFR adaptor protein Shc (Src Homology-2 Domain Containing Transforming Protein) [42]. Shc exists in three isoforms (p46, p52, and p66) and we detected elevated levels of phosphorylated p46 and p52 isoforms upon infection with PAO1 or flagella-coated beads. In contrast, there was no increase in the ratio of phospho-Shc upon addition of the flagella-coated beads to the AP surface or upon AP or BL addition of pili-coated beads. Although, at this point, we are not able to show the activation of signaling events farther downstream of EGFR, such as Akt phosphorylation, these results confirm the significance of EGFR phosphorylation by flagella- but not pili-coated beads.

HB-EGF, but not EGF, needs to bind to HS chains of HSPGs to activate EGFR. Since our results demonstrate that flagella likewise activate EGFR in an HS-dependent manner, we first tested whether flagella-coated beads bind to EGFR or HB-EGF. While flagella-coated beads bound with great avidity to HS coated onto plastic wells, they did not bind measurably to the extracellular domain of EGFR, to HB-EGF, or to EGF, used as a negative control since it does not bind to HS to activate EGFR (data not shown). Second, we tested whether HB-EGF can inhibit binding of flagella-coated beads to HS chains. At a high concentration, exogenous HB-EGF, but not EGF, slightly inhibited binding of flagella-coated beads to HS coated onto plastic wells (Figure S5). These results suggest that, at high concentrations, HB-EGF can either compete with bacterial flagella for binding sites on HS chains or it sterically hinders flagella binding to HS.

Bacterial strains

P. aeruginosa strain O1 (PAO1) was obtained from the ATCC (ATCC 15692) and isogenic mutants PAO1ΔpilA and PAO1ΔfliC were previously constructed in the laboratory [63]. PA103 [64] was a kind gift of Dr. Dara Frank and PA103 Mutant 9 was previously constructed in the laboratory [38]. All strains were routinely grown shaking overnight in Luria-Bertani broth (LB broth) at 37°C. GFP-expressing strains were created by electroporation of the pnpT2-GFP-pUCP20 plasmid as described previously [9].

Bacterial adhesion and invasion assays

Following overnight growth in LB broth at 37° with shaking, bacteria were added to well polarized cells at an MOI of 20. For AP infections, the bacteria in serum-free MEM were added to the AP chamber of cells grown on Transwells. For BL infections, the Transwell insert was placed directly onto 50 µl of serum-free MEM containing bacteria. After 2 h of infection at 37°C, adhesion and invasion assays were performed as described previously [9]. Bacteria were enumerated by plating serial dilutions of cell lysates to LB plates and counting colony-forming units (cfu). All assays were carried out on triplicate wells, and results are reported as the average of three to five experiments.

Isolation of flagella and Tfp

To isolate surface flagella or Tfp, PAO1ΔpilA, PAO1ΔfliC, PA103, or PA103 Mutant 9 were grown shaking overnight in 2 ml LB at 37°C and 100 µl of the culture was plated onto 1.5% LB agar. Following growth at 37°C for 16 h, the bacteria were scraped from the agar surface and resuspended in 5 ml PBS. A volume of cells equivalent to an optical density at 600 nm (OD₆₀₀) of 20.0 was resuspended in 1 ml PBS. Cells were vortexed vigorously at room temperature for 30 min to remove surface flagella or Tfp by shearing. The suspension was centrifuged at 20,000× g for 10 min at 4°C, the supernatant was collected and centrifuged a second time to remove all cellular debris. The resulting supernatant was dialyzed against PBS (pH 7.4) overnight at 4°C and centrifuged at 20,000× g for 20 min at 4°C to remove insoluble proteins. Afterward, the supernatant was incubated overnight at 4°C in 100 mM MgCl₂ to precipitate flagella or Tfp. The precipitate was collected by centrifugation at 4°C (15,000× g for 20 min), and the pellet was resuspended and dialyzed against PBS (pH 7.4) overnight at 4°C. Afterward, the suspension was centrifuged at 20,000× g for 20 min at 4°C to remove insoluble proteins, and flagella or Tfp were precipitated again in 100 mM MgCl₂ at 4°C. Dialysis, centrifugation, and precipitation steps were repeated again to obtain flagella or Tfp of a high purity as assessed by SDS-PAGE and staining in 0.25% Coomassie (Bio-Rad) for 4 h. Destaining was done in 10% ethanol, 7.5% acetic acid for 6 h or until bands appeared and the background was clear.

Preparation of adhesin- or sugar-coated fluorescent beads

For adsorption of bacterial adhesins to beads, 0.5 ml of 2.5% suspension of Green Fluoresbrite Latex fluorescent beads (2 µm ∅; Polysciences Inc.) were mixed with 200 µg of purified Tfp or flagella in 0.1 M Borate Buffer overnight at room temperature according to the manufacturer's protocol. To determine the coating efficiency, coated beads were eluted in SDS sample buffer and analyzed by Western blotting. Following SDS-PAGE and transfer, the membranes were probed with a 1∶100,000 dilution of primary α-FliC (for flagella) or α-PilA (for Tfp) [63] antibody overnight at 4°C, followed by probing with a 1∶25,000 dilution of horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Gels were quantified by densitometry using ChemiDoc XRS and coating efficiencies were calculated. On average, 15–20% (30 µg) flagella and ∼30% (60 µg) Tfp were bound to the beads. For adsorption of glycosylated molecules to beads, 0.5 ml of Red Fluoresbrite Latex fluorescent beads (2 µm ∅; Polysciences, Inc.) were mixed with 400 µg of HS, N-glycan-3, Man, or Gal according to the manufacturer's protocol. Bead coating efficiency (30–40%) was determined by eluting coated beads in SDS sample buffer, dotting an aliquot on a Zeta-Probe membrane (Bio-Rad), staining the membrane with 1% Toluidine Blue, and comparing the staining to standards.

Cell culture

Calu-3 cells were obtained from the ATCC and maintained in MEM supplemented with 10% fetal bovine serum (FBS; Invitrogen) and L-glutamate at 37°C with 5% CO₂. Cells were grown as 2D monolayers on 12-mm Transwell filters (3-µm pore size; Corning Inc.) as previously described [9]. For experiments, Calu-3 cells were seeded at 1.5×10⁶ cells/well and cultured for 3 days (“incompletely polarized monolayers”) or at 1×10⁶ cells/well on Transwells and cultured for 9 days (“well polarized monolayers”). Under each condition, cells formed polarized confluent monolayers as determined by polarized distribution of some AP and BL membrane proteins and the presence of functional tight junctions that were impermeable to small molecules such as FITC-inulin (data not shown and [9]). However, in incompletely polarized monolayers, distribution of HSPGs on the BL surface was not fully polarized [9].

Enzymatic and inhibitory treatments

To remove glycosaminoglycans, Calu-3 cells were treated with 200 mU of heparinase III or chondroitinase ABC (Sigma-Aldrich) in Hank's Buffered Salt Solution (HBSS) containing 0.1% BSA at 37°C for 2 h (resulting in 60–65% reduction in glycosaminoglycan expression), or, for partial removal of glycosaminoglycans, with 50 mU of heparinase III for 30 min (resulting in ∼25% reduction in expression). To assess the efficiency of treatments, HS chains were visualized by IF staining with HS antibody (10E4; Seikagaku), CS chains were visualized with FITC-WFA (CS-specific lectin from Wisteria floribunda; Sigma-Aldrich), and staining densities were quantified using ImageJ and compared to the staining densities of untreated cells (data not shown and [9]). For competition blocking experiments with glycosaminoglycans, cells were pre-incubated with 5 µg/ml of heparin or CS (Sigma-Aldrich) at 37°C for 1 h in serum-free MEM. For up-regulation of N-glycosylation, cells were grown in the presence of 1 mM Man or Gal (Sigma-Aldrich) in MEM with 10% FBS for 1 week (resulting in 1.4-to-1.7 fold increase in N-glycosylation). To inhibit N-glycosylation, cells were pre-treated with 1 µg/ml of tunicamycin (Sigma-Aldrich) for 16 h (∼50% reduction) or for 8 h (brief de-glycosylation resulting in ∼20% reduction) in MEM with 10% FBS. To assess cell surface N-glycosylation, cells were stained with the Man-specific lectin FITC-concanavalin A (Sigma-Aldrich), staining densities were quantified using ImageJ and compared to the staining densities of untreated cells (data not shown and [9]). To inhibit EGFR, PDGFR, or FGFR, cells were pre-incubated with 10 µM AG1478, AG1296, or PD173074 (Calbiochem) in MEM with 10% FBS for 1 h. To inhibit PI3K, cells were pre-incubated with 50 µM LY294002 (Sigma-Aldrich) in MEM with 10% FBS for 1 h. Inhibition efficiencies were quantified by Western blotting using phospho-specific antibodies.

Protein depletion by siRNA

EGFR (sc-29301), PDGFR-α (sc-29443), PDGFR-β (sc-29442), and control (sc-37007) siRNAs were purchased from Santa Cruz Biotechnology. HeLa cells (ATCC CCL-2), grown in MEM supplemented with 10% FBS, were transfected with siRNAs according to the manufacturer's instructions. After 42 h, standard adhesion and invasion assays were performed. In parallel, lysates were immunoblotted with appropriate antibodies to evaluate the efficiency of protein depletion.

In vitro binding assays

HSPGs, glycosaminoglycans, N-glycan-1, -2, and -3, and sugars (Sigma Aldrich; 0.1–10 µg in 0.2 ml ddH₂O) were added to 96-well polystyrene plate (Corning) and incubated overnight at 37°C until evaporated. Wells were washed with ddH₂O and blocked in 0.1% BSA for 0.5 h at room temperature. Bound molecules were stained with 1% Toluidine blue (Sigma-Aldrich) and absorbance was measured at 630 nm. The absorbance of known concentration of molecules was used as the standard curve and the concentration of bound molecules (µg/well) was calculated. 100 µl of flagella- or Tfp-coated beads in ddH₂O were added to coated wells and incubated for 2 h on a rotary shaker. Non-adherent beads were removed by washing with ddH₂O. For some experiments, 5–20 ng/ml HB-EGF or EGFR were added to heparin-coated wells before addition of flagella-coated beads. Bound beads was quantified using a SpectraMax 340PC plate reader using SOFTmaxPro software (Molecular Devices) at λex = 480 nm and λem = 530 nm. Beads bound to non-coated wells were used as a control and subtracted out as background. The results are reported as the average of six experiments, each with at least 6 replicates.

For the bead-bead aggregation assay, red fluorescent beads, 100 µl of green fluorescent beads coated with flagella or Tfp were allowed to aggregate with 100 µl of red fluorescent beads coated with various glycosylated molecules on a rotary shaker at 50 rpm for 2 h in ddH₂O. For competition blocking experiments, 5 µg/ml of heparin or N-glycan-3 (Sigma-Aldrich) were added to wells. Images of aggregates were acquired with a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a 20× objective. Image processing was performed using Adobe Photoshop CS4 version 11.0.2. Quantification of the fraction of green, red, and mixed (yellow) aggregates from 3 separate experiments and 10 events per each sample was performed using UTHSCSA Image Tool version 2.00 Alpha.

Immunoprecipitation and immunoblotting

Well polarized Calu-3 cells grown on Transwells for 9 days were washed and placed in serum-free MEM for ∼17 h. Bacteria at the MOI of 200 or 50 µl adhesin-coated beads were added to the AP or BL chamber for 1 h. As a control, 10 ng/ml HB-EGF was added to the AP or BL-chamber for 10 min. The infected and HB-EGF-treated monolayers were washed with cold PBS containing 1 mM sodium orthovanadate (Sigma-Aldrich). Cells were lysed in modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 0.1 mM okadaic acid (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and proteinase inhibitor tablets (Complete; Roche Diagnostics)) for 20 min, and cells were removed from the Transwell filters by gentle scraping. The cell lysates were centrifuged at 16,000× g for 20 min. Immunoprecipitation with Akt or EGFR antibody (Cell Signaling Technology) using Magnetic Dynabeads Protein G beads (Invitrogen) were performed according to the manufacturer's protocol. For detection of Shc, whole cell lysates were used without immuprecipitation. Cell lysates or eluted immune complexes were separated on Novex-NuPAGE 10% Bis-Tris SDS-PAGE gels (Invitrogen) and electroblotted to iBlot Nitrocellulose Membranes using the iBlot Device (Invitrogen) according to the manufacturer's protocol. Membranes were blocked in PBS containing 0.05% Tween 20 and 5% non-fat milk (PBST) and probed with a 1∶1000 dilution of an antibody that recognizes Akt phosphorylated on serine 473, EGFR phosphorylated on serine 1046/1047, or Shc phosphorylated on tyrosine 239/240 (Cell Signaling Technology) in PBST, overnight at 4°C. Membranes were then incubated with a 1∶3000 dilution of horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) and detected by enhanced chemiluminescence using the Amersham ECL Western blotting detection kit (GE Healthcare). For loading control, membranes were stripped and re-probed with an antibody that recognizes all forms of Akt, EGFR, or Shc (Cell Signaling Technology). Gels were quantified by densitometry using ChemiDoc XRS.

Immunofluorescence microscopy and image analysis

Calu-3 cells grown on Transwells as incompletely polarized monolayers were infected with PAO1ΔpilA-pGFP, PAO1ΔfliC-GFP (MOI 50), or with 50 µl flagella- or Tfp-coated green fluorescent beads for 2 h at room temperature. Afterwards, cells were washed and fixed in PBS containing 1% paraformaldehyde at 37°C for 0.5 h. After washing, cells were incubated with primary antibodies overnight at 4^øC and, afterwards, with fluorescent secondary antibodies for 2 h at room temperature. HS chains were stained with 1∶500 anti-heparan sulfate antibody (10E4; Seikagaku) followed by 1∶2,000 AlexaFluor647-conjugated secondary antibody (Invitrogen). Actin filaments were stained with 1∶2,000 AlexaFluor594-phalloidin (Invitrogen) and Man residues were detected by staining with 1∶1,000 FITC-conjugated lectin concanavalin A (Sigma-Aldrich). Filters were excised and mounted on microscope slides (Fisher Scientific) in mounting medium (Vector Laboratories, Inc.). Samples from 3 separate experiments and 20 events per each sample were examined with a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.). Images and 3D reconstructions were acquired by and processed in Meta 510 software. Image J analysis was performed on TIFF files. Bacterial or bead binding to Calu-3 cells and co-localization with surface markers was quantified using the Image J plugin Voxel counter on 3D reconstructions of TIFF images acquired with Meta 510 software. Voxel Counter (ImageJ plugin) was used to quantify the volume of bound 3D bacterial or bead aggregates and a minimum volume was set as a threshold to enable automated cell counting using the 3D Object Counter (ImageJ plugin). Any aggregate above the threshold was counted as one. The surface area of membrane regions either enriched or depleted of HS or N-glycans (as determined by staining with an anti-HS antibody or with FITC-ConA, respectively) was measured in pixels by ImageJ, and the number of bacterial or bead aggregates bound was normalized per pixel of each specific surface area. The percentage (compared to total) of bacterial or bead aggregates bound to each specific region was determined.

Statistical analysis

Data are expressed as means ± SD (standard deviation). Statistical significance was estimated by ANOVA test using InStat version 3.0b. Differences were considered to be significant at P<0.05.