Listeria monocytogenes Co-Opts the Host Exocyst Complex To Promote Internalin A-Mediated Entry

ABSTRACT

The bacterial pathogen Listeria monocytogenes induces its internalization (entry) into intestinal epithelial cells through interaction of its surface protein, internalin A (InlA), with the human cell-cell adhesion molecule, E-cadherin. While InlA-mediated entry requires bacterial stimulation of actin polymerization, it remains unknown whether additional host processes are manipulated to promote internalization. Here, we show that interaction of InlA with E-cadherin induces the host membrane-trafficking process of polarized exocytosis, which augments uptake of Listeria. Imaging studies revealed that exocytosis is stimulated at sites of InlA-dependent internalization. Experiments inhibiting human N-ethylmaleimide-sensitive factor (NSF) demonstrated that exocytosis is needed for efficient InlA-mediated entry. Polarized exocytosis is mediated by the exocyst complex, which comprises eight proteins, including Sec6, Exo70, and Exo84. We found that Exo70 was recruited to sites of InlA-mediated entry. In addition, depletion of Exo70, Exo84, or Sec6 by RNA interference impaired entry without affecting surface levels of E-cadherin. Similar to binding of InlA to E-cadherin, homophilic interaction of E-cadherin molecules mobilized the exocyst and stimulated exocytosis. Collectively, these results demonstrate that ligation of E-cadherin induces exocytosis that promotes Listeria entry, and they raise the possibility that the exocyst might also control the normal function of E-cadherin in cell-cell adhesion.

INTRODUCTION

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, and abortion (1). Critical for disease is the ability of Listeria to induce its internalization (entry) into nonphagocytic cells in the intestine or placenta (2, 3). This entry process is mediated chiefly by interaction of the Listeria surface protein, internalin (InlA), with its human cell-cell adhesion receptor, E-cadherin (4, 5). Work with animal models indicates that binding of InlA to E-cadherin promotes traversal of the intestinal and placental barriers (3, 6, 7).

Studies with cultured human cells have demonstrated that InlA-mediated entry of Listeria involves bacterial exploitation of the host actin cytoskeleton (8, 9). Interaction of InlA with the extracellular domain (ECD) of E-cadherin induces actin polymerization at sites of bacterial adhesion to host cell plasma membrane (10–13). This localized actin filament assembly is thought to contribute to entry by providing a protrusive force that drives the plasma membrane around adherent bacteria. At least two pathways control actin polymerization during InlA-mediated uptake of Listeria (8, 9). One pathway is mediated by the host Arp2/3 complex and activators of this complex, including the GTPase, Rac1, and the nucleation-promoting factor, cortactin (12). A second pathway involves human factors that normally control endocytosis, including the coat protein, clathrin, and the GTPase, dynamin (13–15).

A critical unresolved question is whether host processes apart from actin polymerization are co-opted by Listeria to promote InlA-mediated entry. One such process could be polarized exocytosis, which is the localized delivery of intracellular vesicles to expand specific sites in the plasma membrane (16). Membrane flow through exocytosis could potentially affect Listeria entry by contributing to remodeling of the host cell surface. Polarized exocytosis is spatially controlled by the exocyst, an octameric protein complex that tethers vesicles to sites in the plasma membrane prior to vesicle-plasma membrane fusion mediated by SNARE proteins (16–18). An endomembrane compartment called the recycling endosome (RE) serves as a source for vesicles for polarized exocytosis (16). Whether InlA-mediated entry of Listeria involves exploitation of the exocyst to stimulate exocytic membrane trafficking and consequent plasma membrane remodeling is presently unknown.

In this study, we demonstrate that the exocyst complex acts downstream of E-cadherin to promote InlA-dependent entry. Using latex beads coupled to InlA, we found that exocytosis is stimulated at sites of InlA-mediated internalization. This local induction of exocytosis was accompanied by recruitment of the exocyst component, Exo70. Impairment of exocytosis through inhibition of N-ethylmaleimide-sensitive factor (NSF) or depletion of exocyst components reduced InlA-dependent entry without significantly affecting clustering or surface levels of E-cadherin. Similar to beads coated with InlA, particles coupled to the extracellular domain (ECD) of E-cadherin induced recruitment of Exo70 and exocytosis. Taken together, our results demonstrate that engagement of E-cadherin mobilizes the exocyst to stimulate the host process of polarized exocytosis. This process enhances Listeria infection and also has the potential to impact normal functions of E-cadherin.

RESULTS

Polarized exocytosis is stimulated during InlA-mediated entry.

As a tool to assess the role of exocytosis in InlA-dependent entry, we used latex beads coated with purified InlA protein. These beads have been extensively used in previous work since they are internalized more efficiently into host cells than Listeria expressing InlA (11, 12, 15, 19–21). In addition, beads coupled to InlA permit selective analysis of the InlA pathway of entry without contributions of alternative Listeria invasion factors, such as the protein InlB (9, 22).

To prepare beads conjugated to InlA, recombinant InlA protein containing an amino-terminal 6× histidine (6×His) tag was expressed in Escherichia coli strain BL21 λDE3 and purified as described (22). After proteolytic removal of the 6×His tag, the resulting protein (see Fig. S1A in the supplemental material) was covalently linked to carboxylate-modified beads (~3 μm in diameter). In order to confirm that beads coupled to InlA are internalized into human cells in an E-cadherin-dependent manner, we used human MCF10A breast cancer cells expressing E-cadherin or an isogenic cell line in which both copies of the E-cadherin gene, CDH1, were inactivated by zinc finger nuclease (ZFN) technology (Fig. S1B) (23). Approximately 40% of InlA-coated beads that bound to wild-type (CD1⁺/CD1⁺) MCF10A cells were internalized, whereas less than 0.30% of the same beads entered into CDH1⁻/CDH1⁻ cells (Fig. S1C). The efficiency of internalization of InlA-coated beads into MCF10A CDH1⁺/CDH1⁺ cells was comparable to that in the human enterocyte cell line, Caco-2 (Fig. S1D), which expresses E-cadherin (Fig. S1B) and is commonly used in studies of InlA (4, 5, 10, 12, 22, 24–27). In contrast to the situation with InlA-coated particles, beads linked to bovine serum albumin (BSA) or glutathione S-transferase (GST) failed to enter into MCF10A (CD1⁺/CD1⁺) or Caco-2 cells (Fig. S1C and D). Collectively, these results verify that beads coupled to InlA are efficiently internalized in a manner dependent on E-cadherin.

We next examined if InlA-coated beads induce polarized exocytosis during entry into human cells. These studies involved Caco-2 cells stably expressing an exocytic probe consisting of the v-SNARE protein VAMP3 fused to enhanced green fluorescent protein (EGFP) (28, 29). This VAMP3-EGFP probe resides in vesicles derived from the RE. When these vesicles fuse with the plasma membrane during exocytosis, the EGFP portion becomes exofacial (extracellular) and can be labeled with antibodies without cell permeabilization (Fig. 1A). In this labeling method, intrinsic EGFP fluorescence represents total cellular VAMP3, and fluorescence due to the EGFP antibody indicates VAMP3 at sites of exocytosis. Importantly, both total and exofacial VAMP3-EGFP accumulated in cuplike structures around InlA-coated beads that were incubated with Caco-2 cells for 15 min (Fig. 1B). As controls for exofacial labeling of VAMP3-EGFP, Caco-2 cells expressing EGFP alone or a probe consisting of EGFP joined to the pleckstrin homology (PH) domain of the kinase Akt were used. Since EGFP is cytoplasmic and the Akt.PH domain localizes to the inner leaflet of the plasma membrane (30), these two proteins should be inaccessible to anti-EGFP antibodies. As expected, no signals were detected for the control proteins by using the exofacial labeling approach (Fig. 1B). The results in Fig. 1B therefore demonstrate that exocytosis occurs in regions of the host cell plasma membrane that contact InlA-coated particles.

FIG 1.

Polarized exocytosis is stimulated during InlA-mediated entry. (A) Method used to detect exocytosis. The VAMP3-EGFP probe resides in intracellular vesicles that fuse with the plasma membrane upon exocytosis. Exofacial (exo) VAMP3-EGFP is detected by incubating human cells with anti-EGFP antibodies in the absence of cell permeabilization. (B) Exocytosis associated with InlA-coated particles. Caco-2 cells expressing the VAMP3-EGFP probe, EGFP alone, or EGFP fused to the PH domain of Akt were incubated with beads coupled to InlA or GST for 15 min. Cells were then labeled for extracellular beads (blue) and exofacial EGFP (exo EGFP) proteins (red) as described in Materials and Methods. Intrinsic EGFP fluorescence (total EGFP) appears green. Arrows indicate beads interacting with the surface of Caco-2 cells. Scale bars, 5 μmol. (C) Quantification of exocytosis. Data are pooled fold enrichment (FE) values of exofacial VAMP3-EGFP from three experiments. Dots represent individual FE values. Horizontal bars are means, and error bars are SD. *, P < 0.05 compared to the VAMP3-EGFP probe in cells incubated with InlA-coated beads. Statistical analysis was performed using ANOVA and the Tukey-Kramer posttest. ND, exocytosis was not detected.

Exofacial VAMP3-EGFP fluorescence around InlA-coated beads generally appeared more intense than fluorescence in areas of the host plasma membrane lacking beads, suggesting that InlA stimulates exocytosis. In order to quantify the degree of accumulation of exofacial VAMP3-EGFP around InlA-coated beads, ImageJ software was used to measure fold enrichment (FE) values as previously described (31–33). FE is defined as the mean fluorescence intensity in a cuplike structure around beads divided by the mean fluorescence intensity throughout the cell (31). FE values greater than 1.0 indicate enrichment of exofacial VAMP3-EGFP and, therefore, an upregulation of exocytosis. The mean FE for ~150 InlA-coated beads was 1.47 (Fig. 1C). In contrast, the mean FE value for control beads coupled to GST, which fail to be internalized (Fig. S1D), was 0.91 (Fig. 1C). Taken together, the results in Fig. 1 demonstrate that exocytosis is locally stimulated during entry of InlA-coated particles.

Host exocytic proteins promote InlA-dependent internalization.

In order to assess if polarized exocytosis contributes to InlA-dependent internalization, we determined if inhibition of human proteins that mediate exocytosis reduces entry of InlA-coated particles. Such human exocytic proteins include members of the exocyst complex and the ATPase N-ethylmaleimide-sensitive factor (NSF) (Fig. S2). The exocyst comprises eight proteins, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (16, 18) (Fig. S2). This complex spatially regulates exocytosis by tethering vesicles derived from the RE to specific sites in the plasma membrane (16, 18). After tethering, the v-SNARE protein VAMP3 on vesicles interacts with t-SNARE proteins on the plasma membrane to stimulate membrane fusion (17). NSF then disassembles SNARE complexes and recycles v-SNAREs for new rounds of exocytosis (34).

We used the cell-permeable fusion peptide TAT-NSF700 (35) to test the role of NSF in entry of InlA-coated beads. This peptide, which has been extensively used as an exocytosis inhibitor (35–40), contains an 11-amino-acid cell-permeable peptide from transactivating regulatory protein (TAT) of HIV-1 joined to a 22-amino-acid peptide from human NSF (35). The TAT moiety allows internalization into mammalian cells, and the NSF700 sequence interferes with endogenous NSF by inhibiting its ATPase activity (35). Importantly, treatment of Caco-2 cells with 10 μM TAT-NSF700 inhibited exocytosis around InlA-coated beads and also internalization of these beads compared to control conditions with 10 μM TAT alone or the vehicle, dimethyl sulfoxide (DMSO) (Fig. 2A to C). Under the same conditions, 10 μM TAT-NSF700 did not affect cell viability (Fig. 2D). These results indicate that host exocytosis has an important role in InlA-mediated entry.

FIG 2.

NSF activity is needed for InlA-induced exocytosis and entry. Caco-2 cells expressing VAMP3-EGFP were left untreated or incubated with the vehicle DMSO, TAT peptide, or TAT-NSF700 peptide for 20 min prior to assessment of exocytosis around InlA-coated beads, entry of beads, and effects on host cell viability. (A) Representative confocal microscopy images of exocytosis around InlA-coated particles. Arrows indicate beads associated with host cells. Exofacial VAMP3-EGFP (exo V3-EGFP) is red, beads are blue, and intrinsic EGFP fluorescence (total V3-EGFP) is green. Scale bars, 5 μmol. (B) Quantification of exocytosis around InlA-coated beads. Each dot in the graph indicates an FE value for a bead. Horizontal bars are means, and error bars are SDs. (C) Effect of TAT-NSF700 treatment on internalization of InlA-coated beads. Data are mean percent internalization values ± SEM. (D) Viability of Caco-2 cells subjected to control conditions or treated with TAT-NSF700. Data are mean relative viability ± SEM. Viability was measured using MTT assays (69) as described in Materials and Methods. *, P < 0.05 compared to the no-treatment or DMSO treatment conditions. Statistical analysis was performed using ANOVA and the Tukey-Kramer posttest.

We next addressed the role of the exocyst complex in InlA-mediated internalization. Since the exocyst is known to target exocytosis to specific sites in the plasma membrane (16, 18), we examined if this complex is mobilized to sites of interaction of InlA-coated beads with host cells. Using an EGFP fusion protein, we found that Exo70 accumulated around InlA-coated beads at 15 min postincubation (Fig. 3), the same time that exocytosis was observed (Fig. 1). As expected, EGFP alone was not enriched around InlA-coated particles. In addition, beads coated with GST failed to recruit Exo70-EGFP. These data indicate that the exocyst is mobilized during InlA-mediated entry.

FIG 3.

Exo70 is recruited to sites of interaction of InlA-coated particles with human cells. Caco-2 cells transiently expressing human Exo70 fused to EGFP (EGFP-Exo70) or EGFP alone were incubated with beads coated with InlA or GST as a control for 15 min. Cells were then fixed and imaged by confocal microscopy. (A) Representative confocal microscopy images. Arrows indicate beads associated with Caco-2 cells. EGFP-tagged proteins are green, and beads are shown in differential interference contrast (DIC) microscopy images. (B) Quantification of accumulation of EGFP-tagged proteins around beads. Data are FE values for EGFP-Exo70 or EGFP alone from three experiments. Horizontal bars are means, and error bars are SDs. *, P < 0.05 as determined by ANOVA and the Tukey-Kramer posttest.

In order to determine if the exocyst has a functional role in InlA-dependent internalization, we used RNA interference (RNAi) to impair expression of several exocyst components. We were able to deplete five of the eight exocyst proteins (Exo70, Exo84, Sec5, Sec6, and Sec8) by transfection of Caco-2 cells with short interfering RNAs (siRNAs) (Fig. 4A). Attempts to deplete Sec3, Sec10, and Sec15 were unsuccessful (data not shown). Importantly, depletion of Exo70, Exo84, and Sec6 impaired internalization of InlA-coated beads into Caco-2 cells (Fig. 4A and B). As expected, knockdown of E-cadherin also reduced entry of these beads. In order to address possible off-target effects of siRNAs on entry, we confirmed that additional siRNAs targeting distinct sequences in exo70, exo84, and sec6 mRNAs impaired internalization of InlA-coated particles (Fig. S3). We also verified that siRNA-mediated depletion of Exo70, Exo84, and Sec6 reduced entry of Listeria (Fig. 4C). For these infection studies, we used a Listeria strain that contains a functional inlA gene but is deleted for a gene encoding the bacterial protein InlB, which mediates an entry pathway distinct from that promoted by InlA (8, 9, 22, 24). Control experiments comparing entry of the inlA⁺ ΔinlB Listeria strain with a strain deleted for both inlA and inlB (ΔinlA ΔinlB) confirmed that the inlA⁺ inlB⁻ strain is internalized into Caco-2 cells through InlA (Fig. 4D). Collectively, the results in Fig. 4 indicate that the exocyst components Sec6, Exo70, and Exo84 contribute to InlA-dependent entry.

FIG 4.

Host Exo70, Exo84, and Sec6 contribute to InlA-dependent entry. Caco-2 cells were transfected with siRNAs against E-cadherin or the exocyst components Exo70, Exo84, Sec5, or Sec8. As controls, cells were mock transfected in the absence of siRNA or transfected with a control, nontargeting siRNA. Cells were then either solubilized for assessment of target protein depletion by Western blotting or incubated with InlA-coated beads or bacteria for measurement of entry. (A) Depletion of target proteins by siRNAs. Left panels show representative Western blots, and graphs display quantified Western blotting data from 3 to 5 experiments. Data are mean relative expression values ± SEM. (B) Effect of siRNAs against E-cadherin or exocyst proteins on entry of InlA-coated beads into Caco-2 cells. The graph shows mean percent internalization values ± SEM from 3 to 5 experiments, depending on the condition. *, P < 0.05 compared to the no-siRNA or control siRNA conditions as determined by ANOVA and the Tukey-Kramer posttest. (C) Effect of siRNAs against E-cadherin or exocyst components on internalization of Listeria. Infection was performed using a bacterial strain that has a functional inlA gene but lacks the gene for inlB (inlA⁺ ΔinlB). Data are mean ± SEM values from 7 experiments. *, P < 0.05 compared the control siRNA condition as assessed by ANOVA and the Tukey-Kramer posttest. (D) Role of InlA in entry of Listeria into Caco-2 cells. Internalization efficiencies of inlA⁺ ΔinlB and ΔinlA ΔinlB strains of Listeria are shown. The data are mean percent internalization values ± SEM from three experiments. *, P < 0.05 as determined by Student’s t test.

In contrast to depletion of Exo70, Exo84, or Sec6, RNAi-mediated knockdown of the exocyst proteins Sec5 or Sec8 failed to affect uptake of InlA-coated beads (Fig. 4A and B). The reason for this lack of effect is unknown but could be related to the observation that Sec5 and Sec8 are expressed at ~5-fold-greater levels than Sec6 in mammalian cells (41). These results suggest that a surplus of free Sec5 and Sec8 may exist outside the exocyst complex. It is therefore possible that the small amounts of Sec5 and Sec8 remaining after siRNA-mediated depletion (Fig. 4A) were sufficient to allow exocyst function.

Inhibition of NSF or exocyst proteins does not affect surface levels or clustering of E-cadherin.

In Drosophila and the mammalian epithelial cell lines Madin-Darby canine kidney (MDCK) and NMuMG, genetic inhibition of the exocyst complex decreases accumulation of E-cadherin in adherens junctions (AJs) (42–45). This effect of exocyst inhibition is thought to reflect a role for the complex in recycling of E-cadherin to the plasma membrane (46). We used a surface protein biotinylation approach (47–50) to investigate if RNAi-mediated depletion of Sec6, Exo70, or Exo84 or inhibition of NSF affects surface levels of E-cadherin in Caco-2 cells. Importantly, treatment of Caco-2 cells with TAT-NSF700 peptide did not impact surface E-cadherin compared to treatment with the TAT peptide control (Fig. 5A). Similarly, siRNA-mediated depletion of Sec6, Exo70, or Exo84 failed to cause statistically significant reductions in surface E-cadherin (Fig. 5B and C). E-cadherin is known to cluster at the cell surface around InlA-coated beads during internalization of these particles into human cells (20, 21). We found that siRNAs targeting Sec6, Exo70, or Exo84 did not reduce E-cadherin clustering around beads coupled to InlA (Fig. 6). Taken together, the results in Fig. 5 and 6 show that the same conditions targeting NSF or exocyst proteins that impair InlA-mediated entry (Fig. 4B and C) fail to reduce surface E-cadherin. Combined with our observations that interaction of E-cadherin with InlA induces exocytosis and recruitment of Exo70 (Fig. 1 and 3), the findings in Fig. 5 and 6 show that the exocyst acts downstream of E-cadherin to promote InlA-dependent entry.

FIG 5.

Inhibition of NSF or depletion of exocyst proteins does not affect surface levels of E-cadherin. (A) Measurement of levels of surface E-cadherin in Caco-2 cells treated with TAT-NSF700. Cells were either left untreated, treated with the vehicle DMSO, or incubated with 10 μM TAT or TAT-NSF700 peptides for 20 min, followed by biotinylation of surface proteins at 4°C using EZ Link Sulfo-NHS-SS-Biotin (31, 33, 48). Cells were then solubilized, and biotinylated proteins were captured using beads coupled to streptavidin-agarose (SA). As a control, lysates were incubated with beads linked to protein A (PA), which fails to capture biotinylated proteins. Biotinylated E-cadherin present in precipitates prepared with SA or PA was detected by Western blotting. The left panel shows representative Western blotting, and the graph contains quantified Western blotting data of mean surface E-cadherin levels ± SEM from three experiments. NS, not significant as determined by ANOVA and the Tukey-Kramer posttest. (B) Measurement of surface E-cadherin levels in Caco-2 cells treated with siRNAs targeting Exo70, Exo84, Sec6, or E-cadherin. After mock transfection in the absence of siRNA or transfection with the indicated siRNAs, surface biotinylation was performed, biotinylated proteins were captured, and biotinylated E-cadherin was detected by Western blotting. A representative Western blot image is shown on the left. The graph contains quantified Western blotting data of mean E-cadherin surface levels ± SEM from 6 experiments. *, P < 0.05 compared to the control siRNA condition as determined by ANOVA and the Tukey-Kramer posttest. NS, not significant compared to the control siRNA condition. (C) Confirmation of depletion of target proteins by siRNAs. Total cell lysates prepared after surface biotinylation were used for Western blotting to detect the indicated exocyst protein or E-cadherin. The images on the left show representative Western blots, whereas the graphs display quantified Western blotting data from 6 experiments. The results are mean relative expression levels ± SEM. *, P < 0.05 compared to the control siRNA condition as determined by ANOVA and the Tukey-Kramer posttest.

FIG 6.

Depletion of exocyst components does not inhibit clustering of E-cadherin during entry. Caco-2 cells were subjected to control conditions or treated with siRNAs against Sec6, Exo70, Exo84, or E-cadherin. Cells were then incubated with beads coated with InlA or BSA for 15 min, followed by fixation in methanol and labeling with antibodies against E-cadherin. (A) Representative confocal microscopy images of cells associated with InlA-coated beads. Panels on the left contain DIC images showing beads, whereas panels on the right display E-cadherin labeling. Arrows indicate positions of beads, and arrowheads point to E-cadherin labeling at cell-cell junctions. (B) Representative images of Caco-2 cells associated with BSA-coated beads. Scale bars in panels A and B indicate 5 μmol. (C) Quantification of accumulation of E-cadherin around beads. Data are FE values from three experiments. Horizontal bars are means and error bars are SD. *, P < 0.05 compared cells incubated with InlA-coated beads in the no-siRNA or control siRNA conditions. NS, not significant. Statistical analysis was performed using ANOVA and the Tukey-Kramer posttest. Note that the mean FE value for E-cadherin was increased by treatment with Sec6 siRNA compared to the control conditions.

Homophilic engagement of E-cadherin stimulates exocytosis and particle internalization.

The ability of InlA to induce exocytosis (Fig. 1) prompted us to investigate if homophilic ligation of E-cadherin has a similar activity. Importantly, latex beads covalently coupled to a protein containing the ECD of human E-cadherin stimulated exocytosis and recruited Exo70 similarly to InlA-coated beads (Fig. 7A and B). In addition, beads linked to the E-cadherin ECD were internalized into Caco-2 cells in a manner dependent on NSF activity and the exocyst proteins Exo70, Exo84, and Sec6 (Fig. 7C and D). Collectively, the results in Fig. 7 show that homophilic engagement of E-cadherin mobilizes the exocyst complex, resulting in an upregulation of exocytosis and remodeling of the plasma membrane, as manifested by particle uptake. These results raise the possibility that the exocyst might act downstream of E-cadherin to mediate some of the normal functions of this cell-cell adhesion receptor.

FIG 7.

Homophilic interaction of E-cadherin stimulates exocytosis and particle internalization. (A) Induction of exocytosis by beads coated with the extracellular domain (ECD) of E-cadherin. Caco-2 cells expressing VAMP3-EGFP were incubated with beads covalently linked to the ECD of E-cadherin (E-cad) or InlA for 15 min, followed by labeling for exofacial VAMP3-EGFP (exo V3-EGFP) and beads. The left panels show representative confocal microscopy images. Beads are blue, exocytosis (exo V3-EGFP) is red, and intrinsic fluorescence of VAMP3-EGFP (total V3-EGFP) is green. Arrows show positions of beads, and scale bars indicate 5 μmol. The graph on the right contains FE values of exo V3-EGFP from three experiments. Horizontal bars are means, and error bars are SDs. NS, not significant as determined by Student’s t test. (B) Mobilization of Exo70 by particles coated with the E-cadherin ECD. Caco-2 cells expressing EGFP-Exo70 or EGFP alone were incubated with beads coated with the E-cadherin (E-cad) ECD or InlA for 15 min, fixed, and imaged by confocal microscopy. Arrows indicate positions of beads. Scale bars, 5 μmol. The graph shows mean FE values ± SDs for EGFP-Exo70 or EGFP from three experiments. Each dot is an FE measurement. *, P < 0.0, as determined by ANOVA and the Tukey-Kramer posttest. NS, not significant. (C) Entry of particles coated with the E-cadherin ECD. Caco-2 cells were incubated with beads coated with the E-cadherin ECD or InlA for 45 min, followed by labeling of extracellular particles and confocal microscopy imaging to assess entry. The graph shows the mean percentage of cell-associated beads that were internalized in three experiments. Error bars are SEM. *, P < 0.05 as determined by Student’s t test. (D) TAT-NSF700 inhibits entry of beads coupled to the E-cadherin ECD. Caco-2 cells were subjected to control conditions or treated with 10 μM TAT or TAT-NSF700 peptides for 45 min, followed by incubation with beads for 45 min to allow particle internalization. The graph shows mean percent internalization values ± SEM from three experiments. *, P < 0.05 as determined by ANOVA and the Tukey-Kramer posttest. (E) siRNAs targeting exocyst proteins or E-cadherin impair internalization of beads coated with the E-cadherin ECD. The graph shown mean percent internalization values ± SEMs from four experiments. *, P < 0.05 compared to the no-siRNA or control siRNA conditions. Statistical analysis was performed using ANOVA and the Tukey-Kramer posttest.

DISCUSSION

In this work, we demonstrated that engagement of E-cadherin by InlA protein of Listeria monocytogenes recruits the exocyst complex to stimulate exocytosis that contributes to internalization of bacteria into human cells. These findings identify exocytosis as a host process which, together with actin polymerization, promotes InlA-dependent uptake.

When targeting exocyst proteins by RNAi, we found that depletion of Sec6, Exo70, and Exo84 impaired entry of InlA-coated beads into Caco-2 cells, whereas depletion of Sec5 and Sec8 did not affect uptake of these particles. In contrast, depletion of Sec5 and Sec8 in Caco-2 cells was reported to inhibit cell-to-cell spread of Listeria, a stage of infection that occurs after entry (28). The extent of knockdown of Sec5 or Sec8 (~80%) was similar in our work on InlA-mediated entry and in the prior work on spread. The previous study showed that the exocyst promotes the elongation of plasma membrane protrusions that mediate the spread of Listeria from an infected human cell to a neighboring cell. While we lack definitive evidence for why depletion of Sec5 and Sec8 affects InlA-mediated entry and cell-to-cell spread of Listeria differently, we offer a speculative explanation. Plasma membrane protrusions that mediate spread of Listeria can attain lengths of ~10 μm, and it was proposed that the exocyst delivers membrane that allows these protrusions to grow (9, 28). If so, then protrusion elongation would be highly dependent on the degree of exocytosis at sites of protrusion formation. In such a scenario, depletion of Sec5 or Sec8 by ~80% might reduce exocytosis to a level that is insufficient for robust spread. In contrast to the long lengths of plasma membrane protrusions with Listeria, InlA-coated beads used in our study are only 3.0 μm in diameter. Less membrane delivery through exocytosis would be expected to be needed for remodeling of the plasma membrane around 3.0-μm particles than for generation of ~10-μm plasma membrane protrusions. This might explain why an ~80% depletion of Sec5 or Sec8, which is expected to reduce but not completely abolish exocytosis, does not affect entry of InlA-coated beads.

Past studies indicate that the host actin polymerization plays an important role in InlA-mediated entry since impairment of actin filament assembly by inhibition of the Arp2/3 complex, the nucleation promotion factor cortactin, and the GTPase Rac1 causes a 60 to 90% reduction in internalization of Listeria or InlA-coated beads into human cells (12). By comparison, we found that depletion of Exo70, Exo84, and Sec6 by RNAi and treatment with TAT-NSF700 peptide result in a 50 to 75% decrease in entry of InlA-coated particles. These results indicate significant roles for both exocytosis and actin polymerization in InlA-dependent uptake and suggest that these two host processes might act together to optimally remodel the host cell plasma membrane during entry. The InlA entry pathway involves the formation of membrane pseudopods that zipper around Listeria and fuse at their tips, resulting in bacterial internalization into phagosomes (5, 8). Actin filament assembly is thought to contribute to pseudopod extension by providing a protrusive force that pushes the plasma membrane around adherent particles (8, 51). By delivering new membrane to pseudopods, polarized exocytosis might enhance the magnitude and/or rate of pseudopod extension. Interestingly, components of the exocyst are known to interact with the Arp2/3 complex or members of the WAVE regulatory complex (52–54). These findings raise the possibility that exocytosis and actin filament assembly might be spatiotemporally coordinated to allow optimal InlA-mediated entry.

An important question is how is the exocyst activated upon binding of InlA to E-cadherin. The cytoplasmic domain of E-cadherin associates with the proteins α-catenin and β-catenin to control cell-cell adhesion (55, 56). Mammalian α- and β-catenin interact (coimmunoprecipitate) with Sec6 and Sec8, suggesting that these catenins might control exocyst function (44). α-Catenin and β-catenin are each crucial for InlA-mediated internalization (10), and it is possible that these catenins recruit the exocyst to E-cadherin to control entry of Listeria. Another protein that couples E-cadherin to the exocyst is type I gamma PI4P 5-kinase (57). This lipid kinase mediates the interaction between E-cadherin and Exo70, thereby mobilizing Exo70 to AJs (45). Future work should determine the extent to which catenins and/or PI4P 5-kinase control the exocyst during InlA-dependent entry.

Several lines of evidence in our studies indicate that E-cadherin acts upstream of the exocyst complex to induce InlA-mediated uptake. First, beads coated with InlA protein induced localized exocytosis and accumulation of the exocyst component Exo70. Second, inhibition of NSF activity or RNAi-mediated depletion of exocyst proteins impaired InlA-mediated entry without reducing surface levels of E-cadherin or clustering of this receptor around beads. In contrast to our findings, previous work has shown that the exocyst can act upstream of E-cadherin to promote recycling of this receptor to the plasma membrane. For example, in Drosophila dorsal thorax epithelial cells, Sec5 and Sec15 promote recycling of E-cadherin to the plasma membrane at AJs (44). Similarly, in Drosophila pupal wing epithelia, cells rearrange to become hexagonally packed through a process that requires endocytosis of E-cadherin and recycling mediated by Sec5 (43). In mammalian cells, Sec8 and the exocyst receptor PAR3 mediate localization of E-cadherin to AJs (42). Collectively, our results, combined with these previous findings, indicate that the exocyst complex has the ability to act upstream to recycle E-cadherin or function downstream of this cell-cell adhesion receptor to remodel the plasma membrane. How these upstream and downstream activities of the exocyst are regulated and possibly coordinated remains unclear.

The normal biological function of E-cadherin is to mediate the formation and maintenance of AJs, which connect epithelial cells into continuous sheets (56–59). This function requires the homophilic interaction of E-cadherin molecules on adjacent epithelial cells, which triggers actin cytoskeletal assembly that promotes the expansion and stabilization of AJs (56, 58, 59). Our results indicate that beads coupled to the purified ECD of E-cadherin resemble InlA-coated beads in their ability to stimulate exocytosis, recruitment of Exo70, and internalization of particles. These results raise the interesting possibility that the exocyst might act downstream of E-cadherin to control the normal activities of this receptor in the formation and/or maintenance of AJs.

The results from this study, combined with previous findings, suggest that the exocyst complex may play a general role in entry of bacterial pathogens into human cells (60). Apart from InlA-dependent internalization, Listeria has a second entry pathway that is mediated by binding of the bacterial surface protein InlB to a human receptor tyrosine kinase called Met (22). Previous work demonstrated that efficient InlB-dependent entry requires localized exocytosis mediated by the exocyst complex (31–33). Moreover, the bacteria Salmonella enterica serovar Typhimurium and Staphylococcus aureus also exploit the exocyst to induce uptake into human cells (61, 62). Future research with additional invasive bacteria should answer whether subversion of the exocyst is a general strategy that bacterial pathogens use to augment their efficiency of infection.

MATERIALS AND METHODS

Bacterial strains, mammalian cell lines, and media.

These studies involved the Listeria monocytogenes strains BUG 1047 (inlA⁺ ΔinlB) and BUG 949 (ΔinlA ΔinlB), which are isogenic with the wild-type strain, EGD (24). BUG 1047 contains an in-frame deletion in the inlB gene and is internalized into mammalian cells in a manner dependent on the Listeria protein, InlA, and its host receptor, E-cadherin (22, 24). BUG 949 has in-frame deletions in both the inlA and inlB genes. BUG 1047 and BUG 949 strains were grown in brain heart infusion (BHI; Difco) broth and prepared for infection as described (63).

The human epithelial cell line Caco-2 subclone BBE1 (ATTC CRL-2102) was grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g of glucose per L and 2 mM glutamine (catalog no. 11995-065; Life Technologies) supplemented with 10 μg per mL of human transferrin (catalog no. T8158; Sigma-Aldrich), penicillin-streptomycin solution, and 10% fetal bovine serum (FBS). A Caco-2 cell line stably expressing VAMP3-EGFP (28, 29) was cultured in the same medium supplemented with 0.50 mg per mL of G418. Cell growth, bacterial infections, and incubations with latex beads were performed at 37°C under 5% CO₂.

Antibodies, inhibitors, purified proteins, and other reagents.

Rabbit antibodies used were anti-InlA (22), anti-E-cadherin (catalog no. ab15148; Abcam), anti-BSA, and anti-GST (catalog no. G7781; Sigma-Aldrich). Mouse monoclonal antibodies used were anti-EGFP (catalog no. 1181460001; Roche), anti-Exo70 (catalog no. D2001; Kerafast), anti-Exo84 (catalog no. sc-5515532; Santa Cruz Biotechnology), anti-Sec3 (catalog no. HPA037706; Sigma-Aldrich), anti-Sec5 (catalog no. ED2002; Kerafast), anti-Sec6 (catalog no. sc-374054; Santa Cruz Biotechnology), anti-Sec8 (catalog no. 610658; Becton Dickenson), anti-Sec10 (catalog no. ab241472; Abcam), anti-Sec15 (catalog no. ED2003; Kerafast), and anti-tubulin (catalog no. T5168; Sigma-Aldrich). Horseradish peroxidase (HRPO)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Secondary antibodies or phalloidin coupled to Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 were obtained from Life Technologies. 6×His-tagged InlA or glutathione S-transferase proteins were expressed in E. coli and purified as previously described (22, 64). After purification of 6×His tagged InlA using His-Bind resin (MerckMillipore) and a HiTrap Q column (GE Healthcare), the 6×His tag was removed by thrombin treatment, as described (22). Latrunculin A (catalog no. L5163) and streptavidin-agarose (catalog no. 85881) were obtained from Sigma-Aldrich. Protein A/G agarose was purchased from Santa Cruz Biotechnology. TAT and TAT-NSF700 peptides were procured from AnaSpec. Purified protein containing the extracellular domain (ECD) of human E-cadherin fused to the Fc region of human IgG1 (10204-H02H) was purchased from Sino Biological. BSA used to couple to carboxylate modified latex beads was from a Pierce bicinchoninic acid (BCA) kit (catalog no. 23225; Thermo Fisher Scientific).

siRNAs.

The sequences of short interfering RNAs (siRNAs) used were 5′-GUGUCAUUGGACAGAUAAAtt-3′ (Exo70), 5′-CAGACAACAUCAAGAAUGAtt-3′ (Exo70-2), 5′-CCAUGAAGGACAUGUUCAAtt-3′ (Exo84), 5′-GAUGGUUUGGCCGUAGUCAtt-3′ (Exo84-2), 5′-GAUUCAGUGAUUUGCGAGAtt-3′ (Sec3), 5′-CAGCUUUCAGUCUGGUCGAtt-3′ (Sec5), 5′-GAAUCCAUAGUCAGUUUAAtt-3′ (Sec6), 5′-AGCCCUGAGCACGCGGAUGtt-3′ (Sec6-2), 5′-AGAACCUGCUUUCAUGCAAuu-3′ (Sec8), 5′-GUUUAAUUUAGGUACUGAUtt-3′ (Sec10), 5′-CAGUUUAACUUAGAUGUCAtt-3′ (Sec15), and 5′-GCUGUAUACACCAUAUUGAtt-3′ (E-cadherin). These siRNAs were obtained from Sigma-Aldrich. The negative nontargeting control siRNA molecule 1 (catalog no. D-001210-01) was purchased from Dharmacon. This siRNA has two or more mismatches with all sequences in the human genome, indicating that it should not target host mRNAs.

Mammalian expression plasmids.

Mammalian expression vectors used were EGFP-C1 (Clontech), pEGFP-C1-Exo70 (65), pPH.Akt-EGFP-N1 (30), and pVAMP3-EGFP-N1 (31, 66).

Transfection.

Caco-2 cells grown in 24-well plates or on 22- by 22-mm coverslips in 6-well plates were transfected with siRNAs using Lipofectamine RNAiMax (Life Technologies) or with plasmid DNA using Lipofectamine 2000 (Life Technologies) as previously described (28). siRNAs were used at a final concentration of 100 nM.

Coupling of proteins to latex beads.

InlA, BSA, and GST proteins were coupled to carboxylate-modified latex beads 3 μm in diameter (Polysciences; catalog no. 09850), using covalent linkage, as described (67).

Western blotting.

For Western blotting of total cell lysates, Caco-2 cells were solubilized in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 0.25% sodium deoxycholate, 0.05% SDS, 50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 10 μg per mL each of aprotinin and leupeptin). Protein concentrations of lysates were determined using a BCA assay kit (Thermo Fisher Scientific), equal protein amounts of each sample were migrated on 8.0, 9.0, or 12.0% SDS-polyacrylamide gels, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Incubation of membranes with primary antibodies or secondary antibodies coupled to horseradish peroxidase and detection using Bio-Rad Clarity Max Western enhanced chemiluminescence (ECL) substrate were performed as described previously (22). Chemiluminescence was detected using an Odyssey imaging system (Li-Cor Biosciences). Bands in Western blotting images were quantified using ImageJ software (version 1.53c) as described (68).

Cell surface biotinylation studies.

For experiments with TAT or TAT-NSF700 peptides, approximately 2.5 × 10⁵ Caco-2 cells were seeded in wells of 6-well plates and grown for about 48 h. Cells were then washed twice in phosphate-buffered saline PBS and incubated for 50 min in DMEM alone, DMEM with 0.1% of the vehicle, dimethyl sulfoxide (DMSO), or DMEM with 10 μM TAT or TAT-NSF700 peptide immediately before biotinylation of surface proteins. In studies involving siRNA transfection, 2.5 × 10⁵ Caco-2 cells were seeded in wells of 6-well plates in the presence of siRNAs and Lipofectamine RNAiMax transfection reagent. Surface biotinylation was performed approximately 72 h after siRNA transfection.

For biotinylation of surface proteins, cells were washed in cold PBS (pH 8.0) containing 1 mM (each) CaCl₂ and MgCl₂ and then incubated at 4°C for 30 min in the same medium with 10 mM of the biotinylating reagent, EZ-Link Sulfo-NHS-SS-Biotin (catalog no. 21331; Thermo Fisher Scientific). Cells were then washed twice and incubated at 4°C for 10 min in PBS (pH 8.0) with 1 mM CaCl₂ and MgCl₂ and 20 mM glycine in order to quench unreacted biotinylation reagent. Cells were then washed and solubilized in lysis buffer (0.5% Triton X-100, 0.5% IGEPAL CA-630, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenyl methyl sulfonyl fluoride [PMSF], and 10 μg per mL each of aprotinin and leupeptin) for 30 min at 4°C. Lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Equal protein amounts of supernatants were incubated with streptavidin-agarose beads or protein A/G agarose as a control for 45 min at 4°C on a rotating wheel. Beads were then washed three times each with lysis buffer and then wash buffer (0.1% Triton X-100, 0.1% IGEPAL CA-630, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10 mM sodium fluoride, 1 mM PMSF, 1 mM sodium orthovanadate, and 10 μg per mL each of aprotinin and leupeptin). Samples were migrated on 7.5% SDS-polyacrylamide gels, transferred to PVDF membranes, and Western blotted with anti-E-cadherin antibodies.

Bacterial entry assays.

Entry of Listeria was measured using gentamicin protection assays in 24-well plates as previously described (22). HeLa cells were infected with Listeria strains BUG 1047 (inlA⁺ ΔinlB) or BUG 949 (ΔinlA ΔinlB) approximately 72 h after transfection with siRNAs. Cells were infected for 1 h in the absence of gentamicin, using a multiplicity of infection of approximately 30:1, and then were incubated in DMEM with 20 μg/mL gentamicin for an additional 2 h. Bacterial internalization efficiencies were expressed as the percentage of the inoculum that survived gentamicin treatment.

Quantification of internalization of beads.

Experiments were performed using Caco-2 cells cultured on 22- by 22-mm coverslips in 6-well plates. In studies involving treatment with TAT or TAT-NSF700 peptides, cells were incubated in serum-free DMEM containing 10 μM each peptide, 0.1% of the vehicle, DMSO, or DMEM alone for 20 min at 37°C in 5% CO₂ prior to addition of beads. Beads coupled to InlA, BSA, GST, or the E-cadherin ECD were added to Caco-2 cells at a ratio of approximately 5 particles to human cells. Cells were centrifuged at 1,000 rpm for 2 min at room temperature to enhance binding of beads and were then incubated for 30 min at 37°C in 5% CO₂ to allow internalization of these particles. Cells were washed in PBS and fixed in PBS containing 3% paraformaldehyde (PFA). Samples were labeled with anti-InlA, anti-GST, anti-BSA, or anti-E-cadherin antibodies and secondary antibodies coupled to Alex Fluor 488 before permeabilization. This approach allows distinction between extracellular and intracellular particles (67). After antibody labeling, cells were permeabilized in PBS containing 0.5% Triton X-100 and incubated with phalloidin coupled to Alexa Fluor 555 for detection of filamentous (F)-actin. Samples were mounted in Molwiol with 1,4-diazabicyclo[2.2.2]octane (DABCO) as an antifade agent. Samples were analyzed using an FV1200 laser scanning confocal microscope equipped with a 1.35-numerical-aperture (NA) oil immersion objective, differential interference contrast (DIC) detection, laser lines of 488 nm, 543 nm, and 633 nm, and photomultiplier tubes for detection of fluorescence. Images from serial sections spaced 1.0 μm apart were used to ensure that all cell-associated beads were detected. Extracellular beads associated with human cells were identified as particles labeled with Alexa Fluor 488, and total beads (intracellular and extracellular) were detected by DIC imaging. Intracellular beads were defined as particles detected by DIC that lacked labeling with Alexa Fluor 488. The data shown in Fig. 2C, Fig. 4B, Fig. 7D and E, and Fig. S3B are from 3 to 8 experiments. In each experiment, at least 100 intracellular beads were scored for the control conditions involving no treatment (Fig. 2C and Fig. 7D) or mock transfection in the absence of siRNA (Fig. 4B, Fig. 7D and E, and Fig. S3B). A similar number of total (intracellular plus extracellular) beads were analyzed for all other conditions. Data were expressed as the percentage of total cell-associated beads that were internalized.

Confocal microscopy analysis.

For analysis of exocytosis in Fig. 1 and 2 and Fig. 7A, subconfluent Caco-2 cells stably expressing VAMP3-EGFP (28, 29) were grown on 22- by 22-mm coverslips and incubated in serum-free DMEM containing beads coated with InlA, GST, or the E-cadherin ECD. A ratio of ~5 particles per human cell was used. Samples were centrifuged at 1,000 rpm for 2 min at room temperature and then incubated for 15 min at 37°C in 5% CO₂. Cells were washed in cold PBS and incubated for 45 min at 4°C with mouse anti-EGFP antibodies (1:50) in DMEM supplemented with 50 mM HEPES (pH 7.5) and 0.5% BSA. This step allowed labeling of exofacial VAMP3-EGFP. Samples were washed three times in cold PBS and then fixed in PBS with 3% PFA for 20 min at room temperature. After quenching of unreacted PFA with 50 mM ammonium chloride, samples were washed twice in PBS and incubated in PBS with 0.5% BSA and anti-mouse antibodies coupled to Alexa Fluor 647. Extracellular beads were labeled by incubation for 45 min at room temperature in PBS with 0.5% BSA containing rabbit antibodies against InlA, GST, or E-cadherin (1:100), followed by washing in PBS and incubation for 45 min in PBS with 0.5% BSA with anti-rabbit antibodies conjugated to Alexa Fluor 405. Cells were washed in PBS and permeabilized in PBS containing 0.4% Triton X-100. F-actin was labeled by incubating samples with phalloidin coupled to Alexa Fluor 555 (1:50) in PBS with 0.5% BSA. Samples were washed in PBS and mounted in Molwiol with DABCO. Control experiments in Fig. 1 were performed similarly to those with VAMP3-EGFP, except that Caco-2 cells were transiently transfected with plasmids expressing EGFP alone or Akt.PH-EGFP.

For experiments assessing recruitment of Exo70 (Fig. 3 and 7B), Caco-2 cells on 22- by 22-mm coverslips were transfected with plasmids expressing EGFP-Exo70 or EGFP alone. Approximately 24 h after addition of plasmid DNA, cells were washed, placed in serum-free DMEM, and incubated for 15 min in serum-free DMEM with InlA- or GST-coated beads, as described above. Cells were then washed twice in PBS and fixed in PBS with 3% PFA. For labeling of endogenous E-cadherin (Fig. 6), cells were fixed by incubation in methanol for 5 min at −20°C. Samples were incubated overnight at 4°C with rabbit anti-E-cadherin antibodies in PBS with 1.0% BSA and 0.1% Tween 20. Samples were washed in PBS and incubated for 45 min at room temperature in PBS 0.5% containing anti-rabbit antibodies conjugated to Alexa Fluor 488.

All samples analyzed by confocal microscopy were mounted in Molwiol supplemented with DABCO. Imaging was performed with an inverted Olympus FV1200 laser scanning confocal microscope, using a 60× 1.35 NA oil immersion objective. Images from serial sections spaced 1.0 μm apart were used to ensure that all cell-associated beads were detected. ImageJ (version 1.53c) software was employed to determine fold enrichment (FE) values for each cell-associated bead. FE is defined as the mean pixel intensity in a ringlike structure around the bead, normalized to the mean pixel intensity throughout the human cell (31–33). The thresholding function of ImageJ was used to measure mean pixel intensities in ringlike structures of exofacial VAMP3-EGFP, Exo70-EGFP, EGFP alone, and endogenous E-cadherin around beads. This function was also used to measure mean pixel intensity throughout the human cell. In each experiment, approximately 50 to 100 extracellular cell-associated beads were analyzed for each condition. The data shown in Fig. 1B, Fig. 2B, Fig. 3B, Fig. 6C, and Fig. 7A and B are pooled FE values from three independent experiments.

Cell viability assays.

Measurement of cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed as previously described (69, 70). In order to obtain relative viability values, absorbance values at 600 nm were normalized to those in cells incubated in DMEM medium lacking DMSO, TAT, or TAT-NSF700 peptides.

Statistical analysis.

Statistical analysis was performed using Prism (version 9.3.1; GraphPad Software). In comparisons of data from three or more conditions, analysis of variance (ANOVA) was used. The Tukey-Kramer test was used as a posttest. For comparisons of two data sets, Student’s t test was used. A P value of 0.05 or lower was considered significant.