Lu/BCAM adhesion glycoprotein is a receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

Abstract

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.

Figure 1. Lu/BCAM is co-precipitated with CNF1 but not with CNFY.

HEK293 (A) or HeLa (B) cells were incubated with GST, GST-CNF1-GST or GST-CNFY-GST as indicated for 20 min at 4°C. Cells were harvested and lysed and the GST proteins together with their bound interaction partners were pulled with anti-GST magnetic beads. Proteins were separated by SDS-PAGE and co-precipitated Lu/BCAM detected by Western-blotting. Note that the major band for Lu/BCAM in the lysate runs at about 67-kDa. The glycosylated, surface exposed protein runs at higher molecular weight. Shown is an example of at least 3 independent experiments.

Figure 2. Competition and colocalization studies with HeLa cells.

GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to HeLa cells. Following 2 h incubation the cells were lysed and the CNF1-catalysed deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting (A). HeLa cells were incubated with an anti Lu/BCAM antibody (AB B12) that binds to the extracellular domain or as control with an anti-Lu/BCAM antibody (AB C16) directed against the intracellular part of the glycoprotein. GST-CNF1 was then added to the cells for 2 h. We followed the toxins uptake by the amount of modified RhoA (shift in SDS-PAGE, B). Shown is a typical result of 3 independent experiments. Colocalization of DyLight488-labeled GST-CNF1 with Lu/BCAM and EEA1 (C) Top: HeLa-cells were treated on ice with DyLight488-labeled GST-CNF1 (5 mg/ml) (green) for 30 min to allow receptor binding. After 30 min cells were transferred to 37° for 30 min to induce uptake. Subsequently cells were fixed and stained for EEA1 (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a maximal projection of all confocal planes. Scale bar indicates 10 µm. Middle: HeLa-cells were treated with labeled GST-CNF1 as in A. After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Bottom: HeLa-cells were treated as in A, but instead of GST-CNF1, cells were treated with DyLight488-labeled GST-CNFY (5 mg/ml) (green). After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Shown is a typical staining of 9 HeLa cells analyzed.

Figure 3. Cells not expressing Lu/BCAM do not respond to CNF1.

A) K562 leukemia cells, which do not express Lu/BCAM (top) and the isogenic cell line K562-Lu/BCAM expressing the receptor (bottom), were treated with GST-CNF1 for different time periods from 1 h to overnight (ON) as indicated. Uptake of the toxin was analyzed by the shift of modified RhoA in SDS-PAGE. B) GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to K562-Lu/BCAM cells for 2 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting. Data are representative for at least 3 independent experiments.

Figure 4. CNF1 directly interacts with Lu/BCAM on the cell surface.

Suspensions of HeLa cells ((A–C) 1×10⁵ cells in 1 ml medium) were incubated for 20 min at 4°C with 2 µg DyLight488-labeled GST-CNF1 (CNF1DL488) alone or together with increasing amounts of rBCAM protein (A). Cells were then washed in PBS and subjected to FACS analysis. The experiment was repeated with 2 µg DyLight488-labeled GST-CNF1 (CNF1DL488) alone or together with increasing amounts of αLu/BCAM antibody (molar ratio CNF∶antibody up to 1∶50, * p<0.05) (B) or a 50-fold excess of the αLRP antibody as indicated (C). Data are presented as % fluorescence of control with CNF1DL488 set to 100%. Non- (D) and Lu/BCAM-expressing (E) K562 cells were either left untreated (mock) or were incubated with 2 µg CNF1DL488 for 20 min at 4°C and, following washing with PBS, subjected to FACS analysis. Results are presented as histogram plots, where single cell events are plotted against cell surface-bound fluorescence (Log FL intensity).

Figure 5. Direct rBCAM-CNF interaction.

A) For dot-blots 5 µl of 3 µM solutions of GST-CNFs, GST-CNF fragments and GST alone, respectively, were spotted onto a nitrocellulose membrane. The membrane was blocked with skimmed milk and recombinant BCAM (6 µM) was added for 1 h at room temperature. Following washing bound rBCAM was detected with an anti-Lu/BCAM antibody. Equal protein load was analyzed by visualizing the GST part of the spotted proteins with an anti GST-antibody. B) Biacore protein-protein interaction studies: An antibody against human IgG (Millipore) was coupled to two lanes of a CM5-biacore chip. As ligand recombinant BCAM containing a C-terminal human IgG domain (Sino biologics) was exclusively guided over lane 2. In a second step, GST-CNF proteins as analyte were guided over both lanes. Bound protein is given as relative units (RU) corrected for the unspecific binding to lane 1 as average plus standard deviation of three independent experiments.

Figure 6. CNF1 binds to the cell surface via a C-terminal peptide.

Suspensions of HeLa cells (1×10⁵ cells in 1 ml medium) were incubated for 20 min at 4°C with 2 µg DyLight488-labeled GST-CNF1 (CNF1DL488) alone, or together with increasing amounts (up to 30 fold) of untagged non-labeled CNF1 and the non-labeled fragments GST-CNF1-(426–1014), GST-CNF1-(720–1014), GST-CNF1-(709–1014) and GST-CNF1-(1–343), respectively. Following washing with PBS, cells were subjected to FACS analysis. Results are presented as mean of cell surface-bound fluorescence (% of control) of three independent experiments+standard deviation.

Figure 7. Summary of the CNF1 domain structure.

CNF1 consists of 1014 amino acids. We found amino acids 1–342 responsible for interaction with the laminin receptor precursor (37LRP). Binding to Lu/BCAM occurs with amino acids 720 to 1014. Data from the literature suggest amino acids 683–730 to mediate binding to mammalian cells indicating this part to be responsible for BCAM binding (white box, RBD). The C-domain represents the catalytic part of the toxin. The two hydrophobic helices (H1 and H2) are crucial for insertion into the endosomal membrane.