Figure 1.
Synthetic AHL molecules used in this study.
(A) N-3-oxo-dodecanoyl-L-homoserine lactone C16H27NO4, MW 297.4 (3O-C12-HSL) which is structurally and functionally identical to those obtained from P. aeruginosa cultures; (B) biotin-conjugated probe N-dodecanoyl-L-homoserine lactone-3-hydrazone-biotin C26H43N5O5S, MW 537.7 (3O-C12-HSL-3H-biotin) and (C) N-dodecanoyl-L-homoserine lactone-biotin C38H41N3O9S, MW 552.7 (3O-C12-HSL-biotin); (D) Fluorescently-tagged probe N-dodecanoyl-L-homoserine lactone-3-hydrazone-fluorescein, C37H40N4O8S, MW 700.8 (3O-C12-HSL-FITC).
Figure 2.
3O-C12-HSL modulates migration of Caco-2 cells in a dose- and time-dependent manner.
Caco-2 cells were cultured in μ-dishes with Ibidi insert until monolayers were confluent, and the insert was removed to get two cell patches with a rectangular cell free gap (500±50 µm width). Cells were treated with 0.018% DMSO (diluent control), or 1.5, 6, 12, 25, 50, 100 and 200 µM 3O-C12-HSL and allowed to migrate. For each dish, 4 images of the migrated cells in the gap area were taken at 0, 24, 48 and 72 h. Migration rates were calculated by measuring the distance between cell monolayer patches (six measurements per image and four images per dish for the each time point) using Image J software. Shown is the mean ± standard errors of at least six independent experiments performed on separate days from different cell passages. Significant differences (* - P≤0.05; ** - P≤0.01;*** - P≤0.001) in mean for migration rate compared with values for cells in the control group as calculated by Student's t test.
Figure 3.
SDS-PAGE analysis of 3O-C12-HSL-3H-biotin complexes from cytoplasmic fraction of Caco-2 cells.
The cytoplasmic fraction was incubated with 0.05 mg 3O-C12-HSL-3H-biotin, 0.05 mg 3O-C12-HSL, and 4 µg biotin or without any additions (as controls). Streptavidin agarose resin-captured complexes were analyzed by SDS-PAGE and subsequently stained with PageBlue protein staining solution. Displayed is a representative gel from one of three independent experiments performed on separate days from different reactions, fraction isolation and cell passages. Indicated bands represent proteins IQGAP1 and 2 respectively identified by in-gel digestion and LC-MS/MS analysis as shown in Table 1. Peptide identification views from MASCOT MS data analyses of IQGAP1 and 2 are shown in supporting information (Dataset S1 and S2).
Table 1.
IQGAP tryptic peptides identified in 3O-C12-HSL-3H-biotin affinity complexes from cytoplasmic fraction of Caco-2 cells using LC-MS/MS.
Figure 4.
IQGAP1 and 3O-C12-HSL interaction analysis using a GST pull-down assay.
(A) Detection of 3O-C12-HSL in the eluates using E.coli JM109 pSB1075 lux reporter bioassay. Eluates from matrix beads were from the following pull-down reaction components: GST-IQGAP1 full-length and 3O-C12-HSL (dashed blue line IQGAP1+3O-C12-HSL); GST-IQGAP1 full-length (red line IQGAP1); 3O-C12-HSL (green line 3O-C12-HSL; GST-actinin-4 full-length and 3O-C12-HSL (lilac line ACTN4+3O-C12-HSL); and without adding (brawn line Matrix alone). As an additional control for the bioassay bacteria reporter in LB medium (not shown) or bacteria reporter in LB medium containing elution buffer, TNGT was used (yellow line E.coli reporter alone). Luminescence was measured during 4.5-h growth. Shown is the mean ± standard errors of at least three independent experiments performed on separate days from different reactions. Significant differences (*) in mean for luminescence compared with values for control groups as calculated by Student's t test. (B) Detection of IQGAP1 and actinin 4 in the eluates, after SDS-PAGE and Western blot analysis. Eluates described in (A) were analyzed here. The data are from one representative of at least three independent experiments.
Figure 5.
3O-C12-HSL modulates phosphorylation of Rac1/Cdc42 and level of IQGAP1.
(A) Caco-2 cells were stimulated with 12, 200 µM 3O-C12-HSL or 0.018% DMSO as diluent control, for 5, 20, 60 min, or 2, 5, 6, 24, 48 h. Total cellular protein extracts were analyzed with Western blot using anti-IQGAP1, 189 kDa (upper panel), anti-phospho-Rac1/Cdc42, 28–25 kDa (middle panel) and anti-GAPDH, 36 kDa for loading control (lower panel). (B) Densitometric analysis. The data are from one representative of at least three independent experiments. Density of bands was normalized against DMSO treated cells; values are median.
Figure 6.
Confocal imaging of IQGAP1 and phosphorylated Rac1/Cdc42 in 3O-C12-HSL-treated Caco-2 cells.
(A) Cell monolayers were stimulated with 1, 12, 200 µM 3O-C12-HSL or 0.018% DMSO as diluent control, for 20 min. Cells were fixed and stained with antibodies against IQGAP1 (red) and phospho-Rac1/Cdc42 (green) and analyzed by confocal laser scanning microscopy. The data are from one representative of at least three independent experiments. Image size is 67.6×67.6 µm and pixel size is 0.13 µm. (B) Quantification of immunofluorescence intensity of IQGAP1 and phospho-Rac1/Cdc42 staining. Columns represent means ± standard error (n = 10). The data are from at least three independent experiments. Significant differences (*) in mean for fluorescence intensity compared with values for control groups as calculated by Student's t test.
Figure 7.
High resolution STED microscopy of IQGAP1 and phosphorylated Rac1/Cdc42 in 3O-C12-HSL-treated Caco-2 cells.
Cell monolayers were stimulated with 12 µM 3O-C12-HSL or 0.018% DMSO as diluent control, for 20 min. Cells were fixed and stained with mouse anti-IQGAP1 and Atto 647N goat anti-mouse antibodies (green) and rabbit anti-phospho-Rac1/Cdc42 and Abberior Star 470SX goat anti-rabbit antibodies (red) and analyzed by confocal and STED microscopy. The data are from one representative of at least two independent experiments. (A) Main panels, bars: 5 µm. (B) Inserts, bar: 5 µm.
Figure 8.
Visualization of 3O-C12-HSL-FITC and IQGAP1 in Caco-2 cells.
(A) Caco-2 cell monolayers were treated with 1 µM 3O-C12-HSL-FITC (green) or 0.018% DMSO as diluent control for 1, 5, 20 or 60 min. Cells were fixed and stained with antibodies against IQGAP1 and Atto 647N goat anti-mouse antibodies (red) and DAPI nucleic acid stain (blue), and were analyzed by confocal laser scanning microscopy, showing an X-Y section (large insert), Z-X section (top) and Z-Y (right). The images are from one representative of at least three independent experiments. Image size is 67.6×67.6 µm and pixel size is 0.13 µm. (B) Measurement of co-localization, based on Pearson's coefficient. Columns show the mean ± standard errors (n = 10) based on three independent experiments. Significant differences (*) in mean for Pearson's coefficient compared with values for control groups as calculated by Student's t test.
Figure 9.
Visualization of 3O-C12-HSL-FITC and F-actin in Caco-2 cells.
(A) Caco-2 cell monolayers were treated with 1 µM C12-HSL-FITC (green) or 0.018% DMSO as diluent control for 1, 5, 20, or 60 min. Cells were fixed and stained with Alexa Fluor 594-conjugated phalloidin to detect F-actin (red) and DAPI nucleic acid stain (blue), and were analyzed by confocal laser scanning microscopy. The images are from one representative of at least three independent experiments. Image size is 67.6×67.6 µm and pixel size is 0.13 µm. (B) Measurement of co-localization, based on Pearson's coefficient. Columns show the mean ± standard errors (n = 10) based on three independent experiments. Significant differences (*) in mean for Pearson's coefficient compared with values for control groups as calculated by Student's t test.
Figure 10.
Model of the communication between P.aeruginosa 3O-C12-HSL and human epithelial Caco-2 cells.
P.aeruginosa 3O-C12-HSL interacts and co-localizes with IQGAP1. The targeting of IQGAP1 by 3O-C12-HSL initiates early event of communication between Caco-2 cells and bacteria via 3O-C12-HSL and can further trigger the essential changes in the cytoskeleton network of epithelial cells. Also, 3O-C12-HSL modulates Caco-2 cell migration in a dose- and time-dependent manner. It also alters the phosphorylation status of Rac1 and Cdc42, and cellular distribution and localization of IQGAP1 from the basolateral to apical side of epithelial cells.