Deglycosylation with neuraminidase reduces <i>S</i>. Enteritidis apical invasion.

Confluent HT29-MTX cells were treated with 250 mU/ml neuraminidase or DPBS for 2h followed by incubation with S. Enteritidis for 1 hour. (A) Immunofluorescence confocal microscopy images of confluent deglycosylated HT29-MTX cells incubated with S. Enteritidis (mCherry, red) stained for MUC1 (214D4, green) and nuclei (DAPI, blue). (B) Immunofluorescence confocal microscopy images of confluent deglycosylated ΔMUC1 cells incubated with S. Enteritidis (mCherry, red) stained for MUC1 (214D4, green) and nuclei (DAPI, blue). (C) Quantification of invaded intracellular S. Enteritidis in confluent HT29-MTX wild type and ΔMUC1 cells with and without neuraminidase treatment. After neuraminidase treatment, cells were incubated with S. Enteritidis for 1h at MOI 15 and subsequently treated with gentamicin (300 μg/ml). Cells were lysed and surviving intracellular bacteria were quantified by colony counts. Invasion is expressed as percentage of initial inoculum. Values are the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was performed by Student’s t-test using GraphPad Prism software. * p<0.05; ns, not significant. White scale bars represent 20 μm.</p

The <i>Salmonella</i> SiiE adhesin is responsible for MUC1-mediated invasion.

(A) Immunofluorescence confocal microscopy imaging of confluent HT29-MTX cells infected with S. Enteritidis wild type and siiE knockout bacteria (mCherry, red) stained for MUC1 (214D4, green) and nuclei (DAPI, blue). (B) Immunofluorescence confocal microscopy imaging as above with non-confluent HT29-MTX cells. (C) Quantification of invaded intracellular S. Enteritidis wild type and siiE knockout bacteria. Confluent HT29-MTX cells were incubated with S. Enteritidis for 1h at MOI 15 and subsequently treated with gentamicin (300 μg/ml). Cells were lysed and surviving intracellular bacteria were quantified by colony counts. Invasion is expressed as percentage of initial inoculum. Values are the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was performed by Student’s t-test using GraphPad Prism software. * p<0.05; ns, not significant. White scale bars represent 20 μm.</p

MUC1 expression and <i>Salmonella</i> invasion in different intestinal epithelial cell lines.

(A) Western blot analysis of MUC1 expression levels in 5-day grown early-confluent intestinal epithelial cell lines incubated with or without the pro-inflammatory cytokine IL-6 at 100 ng/ml for 24h. (B) Immunofluorescence confocal microscopy imaging of 5-day grown early-confluent intestinal epithelial cell lines HT29-MTX, HT-29, Caco-2 and HRT-18 cells incubated with S. Enteritidis (mCherry, red) at MOI 60 for 1h stained with anti-MUC1 214D4 antibody (green) and DAPI (blue). White scale bars represent 20 μm.</p

Schematic model showing the SiiE-MUC1 apical invasion pathway.

(A) MUC1 is expressed on the apical surface of intestinal epithelial cells. Salmonella breaches the soluble mucus layer (green) and uses the giant adhesin SiiE to attach to MUC1 and invade the epithelial cells. After MUC1-SiiE-mediated invasion, large SiiE-negative Salmonella clusters are observed in the infected cells. (B) Hypothetical structural model of the SiiE-MUC1 interaction during Salmonella apical invasion of intestinal epithelial cells based on the molecular sizes of the involved players. The needle-like type III secretion system (T3SS) injectosome is essential for Salmonella invasion but is only 80 nm in length. The large transmembrane mucin MUC1 extends 200 nm from the cell surface and contains an extracellular domain with 42 tandem repeats that are highly decorated with O-linked glycans with terminal sialic acids. The giant adhesion SiiE is 175 nm in size and secreted through the type I secretion system (T1SS). SiiE contains 53 Blg repeats with sugar-binding capacity. Our data show that MUC1, sialic acids and SiiE are essential in mediating apical entry of Salmonella. We hypothesize that the SiiE-MUC1 interaction positions the Salmonella T3SS needle close enough to the host membrane to inject its virulence factors and induce uptake.</p

Knockout of MUC1 reduces <i>S</i>. Enteritidis apical invasion.

(A) Schematic representation of the MUC1 coding region with different domains and the position of the guide RNAs (gRNAs) to generate a genetic deletion. SP, signal peptide; VNTR, variable numbers of tandem repeats; SEA, sea urchin sperm enterokinase agrin domain; TM, transmembrane domain; CT, cytoplasmic tail. The position of the deletion confirmation PCR primers is indicated. (B) Confirmation PCR of wild type HT29-MTX (WT) and HT29-MTX MUC1 knockout cells (ΔMUC1). (C) Western blot analysis of HT29-MTX wild type (WT) and MUC1 knockout cells (ΔMUC1) stained for MUC1 (214D4 antibody) or Actin. (D) Immunofluorescence confocal microscopy imaging showing apical invasion of confluent HT29-MTX wild type and ΔMUC1 cells after S. Enteritidis infection (1h) at MOI 60. MUC1 antibody 214D4 was used to stain MUC1 (green) and DAPI was used to stain nuclei (blue). (E) Quantification of S. Enteritidis invasion into confluent HT29-MTX wild type and ΔMUC1 cells. Confluent cells were incubated with S. Enteritidis for 1h at MOI 15 and subsequently treated with gentamicin (300 μg/ml). Cells were lysed and surviving intracellular bacteria were quantified by colony counts. Invasion is expressed as percentage of initial inoculum. Values are the mean ± SEM of three independent experiments performed in triplicate. (F) Immunofluorescence confocal microscopy imaging showing invasion of non-confluent HT29-MTX wild type and ΔMUC1 cells after S. Enteritidis infection as described under A. (G) Quantification of S. Enteritidis invasion into non-confluent HT29-MTX wild type and ΔMUC1 cells as described under E. Statistical analysis was performed by Student’s t-test using GraphPad Prism software. * pp<0.01; ns, not significant. White scale bars represent 20 μm.</p

MUC1 is a receptor for the <i>Salmonella</i> SiiE adhesin that enables apical invasion into enterocytes - Fig 6

SiiE-positive Salmonella closely associate with MUC1 at the apical surface while invaded Salmonella are negative for SiiE (A) Immunofluorescence confocal microscopy imaging of confluent HT29-MTX cells infected with S. Enteritidis wild type bacteria (mCherry, red) stained for SiiE (polyclonal anti-SiiE, green), MUC1 (214D4, white) and nuclei (DAPI, blue). (B) Immunofluorescence confocal microscopy imaging detail showing localization of SiiE-positive bacteria (mCherry and green) on MUC1-positive cup-like structures (white). Bacteria inside the cup are negative for SiiE. (C) 3D projection of confocal microscopy image depicted in A. (D) Quantification and localization of SiiE-positive spots (green) and Salmonella-positive spots (red) relative to the MUC1-positive cellular surface using IMARIS software. White scale bars represent 20 μm.</p

<i>Salmonella</i> invades intestinal HT29-MTX cells that express MUC1.

(A) Immunofluorescence confocal microscopy imaging of confluent HT29-MTX cells with anti-MUC1 antibody 214D4 (green) and DAPI to stain the nuclei (blue). (B, C) Immunofluorescence confocal microscopy infection experiment with confluent HT29-MTX cells and Salmonella enterica Enteritidis (mCherry, red) at MOI 60 for 1 hour stained with anti-MUC1 214D4 antibody (green) and DAPI to stain the nuclei (blue). (D) Infection experiment with confluent HT29-MTX cells and Salmonella enterica Typhimurium (GFP, green) at MOI 60 for 1 hour stained with anti-MUC1 214D4 antibody (red) and DAPI (blue). White scale bars represent 20 μm.</p