Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jan;77(1):517-31.
doi: 10.1128/IAI.00695-08. Epub 2008 Nov 17.

Human decay-accelerating factor and CEACAM receptor-mediated internalization and intracellular lifestyle of Afa/Dr diffusely adhering Escherichia coli in epithelial cells

Julie Guignot  1 , Sylvie Hudault, Imad Kansau, Ingrid Chau, Alain L Servin
Affiliations

Human decay-accelerating factor and CEACAM receptor-mediated internalization and intracellular lifestyle of Afa/Dr diffusely adhering Escherichia coli in epithelial cells

Julie Guignot et al. Infect Immun. 2009 Jan.

Abstract

We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the alpha5 or beta1 integrin subunits nor alpha5beta1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
No cell association and internalization of Dr or AfaE-III fimbria-positive E. coli within cells engineered for expression of α5β1 integrin. Cells were infected with recombinant E. coli strains (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. Cell-associated and internalized bacteria were determined as described in Materials and Methods. (A) Western blot analysis using MAb anti-β1 integrin subunit (clone MAR4) of total cell extracts of untransfected CHO and transfected CHO-α5β1 cells showing the absence of β1 integrin in the CHO cells and its presence in the transfected cells. PAb anti-actin was used to demonstrate equal gel loadings (left micrographs). Transfection of CHO for α5β1 integrin expression renders the cells adhering onto fibronectin but not onto collagen (right micrographs). (B) Cell-associated and internalized bacteria in CHO and CHO-α5β1 cells infected with strain AAEC185, AAEC185DrD+E+, or AAEC185Afa-IIID+E+. (C) HeLa cells were untransfected or transfected with INTERFERin alone or with two siRNAs targeting β1 integrin (β1-1 or β1-2). Western immunoblot analysis using the MAb anti-β1 integrin subunit (clone MAR4) shows the presence of β1 integrin in untransfected cells and INTERFERin-transfected cells; no β1 integrin was found in transfected siRNAs β1-1 or β1-2 cells. PAb anti-tubulin was used to demonstrate equal gel loadings. Untransfected and transfected cells were infected with AAEC185DrD+E+ bacteria. The levels of cell-associated and internalized bacteria (CFU/5 × 105 cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments carried out in triplicate. Statistical analysis was conducted with a global F test. *, P < 0.01.
FIG. 2.
FIG. 2.
Cell association and internalization of DrD+E+ fimbria-positive E. coli within CHO cells engineered to express DAF, CEACAM1, CEA, or CEACAM6 alone and to coexpress α5 or β1 integrin subunits. Cells were infected with AAEC185, WT-IH11128, or AAEC185DrD+E+ bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128, the infection assay was conducted in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. Cell-associated and internalized bacteria were determined as described in Materials and Methods. (A) Western blot analysis using MAb D14HD11 anti-CEACAMs or MAb 8D11 anti-DAF of total cell extracts of CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells showing the absence of DAF and CEACAMs in CHO cells and the presence of DAF in CHO-DAF cells, CEACAM1 in CHO-CEACAM1, CEA in CHO-CEA, and CEACAM6 in CHO-CEACAM6-transfected cells. PAb anti-actin was used to demonstrate equal gel loadings. (B) Cell-associated and internalized bacteria in CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells infected with AAEC185, WT-IH11128, or recombinant AAEC185DrD+E+ strains. (C) Cell-associated and internalized bacteria in CHO-CEACAM1, CHO-CEACAM1 Y493F, CEACAM1 Y520F, and CEACAM1 ΔCT cells infected with the recombinant AAEC185DrD+E+ strain. (D) Cell-associated and internalized bacteria in CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells cotransfected with α5 or β1 integrin subunits and infected with the recombinant AAEC185DrD+E+ strain. In these cells, the expression of the α5 or β1 integrin subunit has been controlled by Western blot analysis (not shown). In addition, when the α5 or β1 integrin subunit was expressed in CHO cells transfected with DAF or the CEACAMs, no change in the distribution of DAF or the CEACAMs was observed (not shown). The levels of cell-associated and internalized bacteria (CFU/5 × 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. In panel B, statistical analysis was conducted with a global F test. P was <0.01 with WT-IH11128- or AAEC185DrD+E+-infected CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells compared with AAEC185-infected cells.
FIG. 3.
FIG. 3.
Microtubules and lipid rafts but not F-actin microfilaments were involved in internalization of AAEC185DrD+E+ bacteria within CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells. Cells were infected with E. coli (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. Treatments with disrupting agents are described in Materials and Methods. The levels of cell-associated (B) and internalized (A) bacteria (CFU/5 × 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. In panels A and B, statistical analysis was conducted with a global F test. *, P < 0.01 for treated cells versus untreated cells.
FIG. 4.
FIG. 4.
DraE and AfaE-III adhesin subunits are sufficient to promote the internalization of WT-IH11128, AAEC185Dr, and AAEC185Afa-III bacteria within CHO-DAF-α5β1 cells. CHO-DAF-α5β1 cells were infected with WT-IH11128D+E+, WT-IH11128D−E+, AAEC185DrD+E+, AAEC185DrD−E+, AAEC185Afa-IIID+E+, AAEC185Afa-IIID−E+, AAEC185Afa-IIID+E−, or AAEC185Afa-IIID−E− bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D−E+, the infection assay was conducted in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. Cell-associated and internalized bacteria were determined as described in Materials and Methods. The levels of cell-associated and internalized bacteria (CFU/5 × 105 DAF-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. *, P < 0.01 versus AAEC185.
FIG. 5.
FIG. 5.
DraE and AfaE-III adhesin subunits are sufficient to promote the clustering of β1 integrin subunits around adhering bacteria. CHO-DAF-α5β1 cells were infected with WT-IH11128D+E+, WT-IH11128D−E+, AAEC185DrD+E+, AAEC185DrD−E+, AAEC185Afa-IIID+E+, or AAEC185Afa-IIID−E+ bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D−E+, the infection assay was conducted in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. Nonpermeabilized cells were fixed and coimmunolabeled as described in Materials and Methods to detect β1 integrin (red) using MAb anti-β1 integrin (clone P5D2) and adhering bacteria using PAb anti-Dr adhesin (green). Arrows indicate the bacteria decorated by positive β1 integrin immunolabeling. Merged images show the colocalization of β1 integrin and adhering bacteria. Micrographs are representative of three experiments.
FIG. 6.
FIG. 6.
Intracellular survival of WT-IH11128 and AAEC185DrD+E+ bacteria in CHO-DAF, -CEACAM1, -CEA, -CEACAM6, and HeLa cells. Cells were infected (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D−E+, the infection assay was conducted in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. The infected cells were then incubated for 1 h in a medium containing 100 μg/ml of gentamicin to kill the extracellular adhering bacteria. The infected cells were subsequently cultured in the presence of cell culture medium containing gentamicin (15 μg/ml) to prevent replication of any remaining viable extracellular bacteria. Internalized bacteria were determined 24, 48, 72, and 96 h postinfection as described in Materials and Methods. The levels of internalized bacteria (CFU/5 × 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. (A) Intracellular survival of WT-IH11128 bacteria in CHO-DAF, -CEACAM1, -CEA, and -CEACAM6 cells. At 96 h postinfection, Student's t test determined a P value of <0.01 for CHO-CEACAM1 and CHO-CEACAM6 cells and a P value of <0.05 for CHO-CEA cells versus CHO-DAF cells. (B) Comparison of rates of intracellular survival of WT-IH11128 and AAEC185DrD+E+ bacteria in HeLa cells. Student's t test determined a P value of <0.01 (72 h postinfection) for AAEC185DrD+E+ and a P value of <0.05 (96 h postinfection) for WT-IH11128.
FIG. 7.
FIG. 7.
Localization of WT-IH11128 bacteria within infected HeLa cells 24 h postinfection shows WT-IH11128-containing vacuoles positive for Lamp-1, Lamp-2, CD63, and LysoTracker but not for DAF or cathepsin D. Cells were infected with GFP-WT-IH11128 bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. The infected cells were treated and subcultured with gentamicin to prevent extracellular bacterial growth as described in the legend to Fig. 6. Cells were fixed and coimmunolabeled as described in Materials and Methods to detect markers, intracellular and extracellular bacteria, DAF, Lamp-1, Lamp-2, CD63, cathepsin D, or LysoTracker (blue). Intracellular bacteria are green labeled, and extracellular bacteria are green and red labeled. Merged images show the colocalization of Lamp-1, Lamp-2, and CD63 (blue) and intracellularly localized bacteria (green). No colocalization was seen of DAF and cathepsin D (blue) and intracellularly localized bacteria (green). For LysoTracker, a merged close-up image shows the colocalization of LysoTracker fluorescence (blue) and intracellularly localized bacteria (green). Arrows indicate the colocalization or lack of colocalization of intracellular bacteria and markers. Micrographs are representative of at least three experiments.
FIG. 8.
FIG. 8.
WT-IH11128-containing vacuoles do not interact with the autophagic pathway. HeLa-GFP-LC3 cells stably transfected for the expression of the GFP-labeled autophagosomal marker, GFP-LC3, were infected with WT-IH11128 bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air in the presence of 1% d-mannose to prevent type 1-pilus-mediated binding. The infected cells were treated and subcultured with gentamicin to prevent extracellular bacterial growth as described in the legend to Fig. 6. (A) Cells were fixed and immunolabeled as described in Materials and Methods for the detection of intracellular bacteria (green) and extracellular bacteria (green and red). Merged close-up images show that there was no colocalization of GFP-LC3 (blue) and intracellular (green) bacteria. Micrographs are representative of three experiments. (B) HeLa-GFP-LC3 cells were treated with rapamycin (500 nM) to activate autophagy. Micrographs show that punctate GFP-LC3 was greater in rapamycin-treated cells than in untreated cells. (C) The graph shows that the intracellular survival of WT-IH11128 bacteria was the same in rapamycin-treated cells as in untreated cells. The levels of internalized bacteria (CFU/5 × 105 DAF-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson, K. L., J. Billington, D. Pettigrew, E. Cota, P. Simpson, P. Roversi, H. A. Chen, P. Urvil, L. du Merle, P. N. Barlow, M. E. Medof, R. A. Smith, B. Nowicki, C. Le Bouguenec, S. M. Lea, and S. Matthews. 2004. An atomic resolution model for assembly, architecture, and function of the Dr adhesins. Mol. Cell 15647-657. - PubMed
    1. Bampton, E. T., C. G. Goemans, D. Niranjan, N. Mizushima, and A. M. Tolkovsky. 2005. The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1 23-36. - PubMed
    1. Beauchemin, N., P. Draber, G. Dveksler, P. Gold, S. Gray-Owen, F. Grunert, S. Hammarstrom, K. V. Holmes, A. Karlsson, M. Kuroki, S. H. Lin, L. Lucka, S. M. Najjar, M. Neumaier, B. Obrink, J. E. Shively, K. M. Skubitz, C. P. Stanners, P. Thomas, J. A. Thompson, M. Virji, S. von Kleist, C. Wagener, S. Watt, and W. Zimmermann. 1999. Redefined nomenclature for members of the carcinoembryonic antigen family. Exp. Cell Res. 252243-249. - PubMed
    1. Berger, C. N., O. Billker, T. F. Meyer, A. L. Servin, and I. Kansau. 2004. Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC). Mol. Microbiol. 52963-983. - PubMed
    1. Billker, O., A. Popp, V. Brinkmann, G. Wenig, J. Schneider, E. Caron, and T. F. Meyer. 2002. Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways. EMBO J. 21560-571. - PMC - PubMed

Publication types

LinkOut - more resources

-