PI3K inhibition decreases bacterial uptake via CEACAM1 and CEACAM1-ΔCT.

<p>(<b>A</b>) 293 cells were co-transfected with mKate-tagged CEACAM1 lacking the cytoplasmic domain (CEACAM1-ΔCT) together with GFP-tagged Btk-PH domain and infected or not with Pacific Blue labelled Opa_CEA-expressing gonococci for 60 min. After fixation, samples were analyzed by confocal microscopy. Arrowhead highlights CEACAM1-ΔCT clustering by gonococci and recruitment of Btk-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm. (<b>B</b>) 293 cells were transfected with the empty vector (pcDNA), HA-tagged CEACAM1 wildtype or CEACAM1-ΔCT. CEACAM expression was analysed in whole cell lysates (WCLs) by Western blotting with α-HA antibody (upper panels) and equal loading of the samples was demonstrated by α-tubulin antibody (lower panel). (<b>C and D</b>) Cells transfected as in (B) were pretreated for 30 min with 50 µM of PI3K inhibitor LY294002 (LY) or left untreated. Cells were infected for 2 h with Opa_CEA-expressing gonococci and total cell-associated (C) or viable intracellular bacteria (D) were quantified. Bars represent mean values ± S.E.M of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without PI3K inhibitor treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001.</p

Synthetic Glycosphingolipids for Live-Cell Labeling

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part ormore commonlyto the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C₄ to C₂₀). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes

Extracellular Ig_C2-like domains of CEACAM1 are required for the PI3K dependency during pathogen uptake.

<p>(<b>A</b>) Domain organization of the used CEACAM constructs. N – Ig_V-like N-terminal domain; A1, B, A2 – Ig_C2-like domains. (<b>B</b>) 293 cells were transfected with HA-tagged CEACAM1, CEACAM1-N, CEACAM1NA1 or CEACAM1NA1B. CEACAM expression was confirmed by Western blotting with α-HA antibody (upper panel). Equal loading of the samples was verified by α-tubulin blot (lower panel). (<b>C</b>) Cells transfected as in (B) were pretreated for 30 min with 200 nM wortmannin (WM) or left untreated before infection with fluorescein-labelled Opa_CEA-expressing gonococci for 2 h. Infected cells were analysed by flow cytometry and the fluorescein signal from internalized bacteria was detected in the presence of trypan blue, which quenches fluorescence derived from extracellular gonococci. Bars represent mean values ± S.E.M from three independent experiments. Numbers are expressed relative to cells expressing CEACAM1 without WM treatment.</p

Bacterial uptake via several epithelial CEACAMs requires PI3K activity.

<p>(<b>A</b>) 293 cells were transfected with empty vector (pcDNA) or constructs encoding CEACAM1, CEA, or CEACAM6. CEACAM expression was confirmed by Western blotting of whole cell lysates (WCLs) with α-CEACAM antibody (upper panel). Equal loading of the samples was verified by α-tubulin blot (lower panel). (<b>B, C</b>) Cells transfected as in (A) were pretreated for 30 min with 200 nM wortmannin (WM) or left untreated. After infection for 2 h with Opa_CEA-expressing gonococci, total cell-associated bacteria (B) or recovered intracellular bacteria (C) were quantified. Bars represent mean ± SEM of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without WM treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001.</p

PI(3,4,5)P and PI(4,5)P are generated during CEACAM1- and 3- mediated bacterial entry.

<p>(<b>A</b>) 293 cells were cotransfected with constructs encoding mKate-tagged CEACAM1-4L or CEACAM3 together with GFP-tagged Btk-PH domain (which binds specifically to PI(3,4,5)P). Cells were left uninfected or were infected with Pacific Blue labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> for 60 min, fixed and analysed by confocal microscopy. Arrowheads highlight bacteria associated with CEACAMs and the Btk-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm. (<b>B</b>) Cells were cotransfected with CEACAM constructs as in A) together with the GFP-tagged PLC-δ-PH domain (which specifically binds to PI(4,5)P). Cells were infected and analyzed as in (A). Arrowheads highlight bacteria associated with CEACAMs and the PLC-δ-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm.</p

Constitutive active PI3K increases, whereas expression of SHIP decreases uptake of Opa_CEA-expressing gonococci via CEACAM1.

<p>(<b>A</b>) 293 cells were cotransfected with constructs encoding CEACAM1-HA or CEACAM3-HA together with cerulean-tagged PI3K or cerulean alone. Cells were infected with fluorescein-labelled Opa_CEA- expressing gonococci for 2 h. Samples were analysed using a flow cytometer by gating on cerulean-positive cells. Dot blot shows a representative gate used to detect the cerulean-positive cell population. (<b>B</b>) The fluorescein signal derived from cerulean-positive cells in (A) was quantified in the presence of trypan blue, which quenches fluorescence derived from extracellular gonococci. Bars represent mean values ± S.E.M of a representative experiment done in triplicate. (<b>C–E</b>) 293 cells were cotransfected with CEACAM1-HA or CEACAM3-HA together with cerulean, cerulean-tagged SHIP1 phosphatase domain (SHIP), or an inactive form of the SHIP1 phosphatase domain (SHIP D675G), respectively. (C) Expression of cerulean, SHIP1, or SHIP1 D675G was analysed by flow cytometry and histograms show representative samples. (D and E) Cells were infected with fluorescein-stained Opa_CEA-expressing gonococci for 2 h and the fluorescein signal derived from cerulean-positive cells was detected in the absence (D) or presence (E) of trypan blue. This allows quantification of total cell-associated bacteria (D) or internalized gonococci (E). Bars represent mean values ± S.E.M of three independent experiments. Numbers are expressed relative to cells co-expressing cerulean and the respective receptor.</p

CEACAM1-mediated uptake of Opa_CEA-expressing <i>N. meningitidis</i> by endothelial cells depends on PI3K activity.

<p>(<b>A</b>) Human brain microvascular endothelial cells (HBMEC) were transduced with GFP-encoding control lentivirus (control) or a CEACAM1-GFP-encoding lentivirus. CEACAM1 expression in transduced HBMEC is analysed by Western blotting of whole cell lysates (WCL) using monoclonal α-CEACAM antibody (upper panel) and equal loading of the samples was demonstrated by α- tubulin antibody (lower panel). (<b>B</b>) Stable CEACAM1-GFP or control GFP expressing HBMECs were infected with biotin- and AlexaFluor647-labelled Opa_CEA-expressing meningococci for 60 min. Upon fixation and and staining of extracellular bacteria with streptavidin-rhodamine, samples were analyzed by confocal microscopy. Arrows highlight intracellular bacteria, whereas arrowheads point to extracellular bacteria. Bars represent 5 µm. (<b>C and D</b>) CEACAM1-GFP or GFP expressing HBMECs were pretreated with the indicated concentrations of wortmannin for 30 min or left untreated. Cells were infected for 3 h with Opa_CEA-expressing meningococci and total cell-associated (C) or viable intracellular bacteria (D) were quantified. Bars represent mean values ± S.E.M of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to CEACAM1-GFP cells without PI3K inhibitor treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001, *, p<0.05.</p

Inhibition of PI3K activity decreases uptake of Opa_CEA-expressing gonococci via CEACAM1.

<p>(<b>A</b>) 293 cells were transfected with empty vector pcDNA, CEACAM1-4L or CEACAM3 WT. CEACAM expression was verified by Western blotting of whole cell lysates (WCL) using α-HA antibody. (<b>B</b>) Cells were transfected as in (A) and pretreated for 30 min with 50 µM of the PI3K inhibitor LY294002 (LY). After infection for 2 h with Opa_CEA-expressing gonococci, the number of total cell-associated bacteria was determined. Bars represent mean values ± S.E.M of three independent experiments done in triplicate. Total cell associated bacteria are shown relative to cells expressing the respective receptor without PI3K inhibitor treatment. (<b>C</b>) Cells were transfected and infected as in (B). Viable intracellular bacteria were determined in gentamicin protection assays. Bars represent mean values ± S.E.M of three independent experiments done in triplicate. Recovered bacteria are shown relative to cells expressing the respective receptor without PI3K inhibitor treatment. (<b>D</b>) 293 cells were cotransfected with mKate-tagged CEACAM1 and the GFP-tagged Btk-PH domain. 30 min before infection with Pacific Blue labelled Opa_CEA-expressing gonococci, cells were treated with 200 nM wortmannin or left untreated. Fixed samples were analyzed by confocal microscopy. Arrowheads highlight CEACAM- recruitment to cell-associated bacteria. Insets show enlargement of the highlighted area. Bars represent 5 µm.</p

Overexpression of a CEACAM8/1 chimera interferes with CEACAM1-mediated uptake.

<p>(<b>A</b>) Domain organization of the used CEACAM constructs. N – Ig_V-like N-terminal domain; A1, B, A2 – Ig_C2-like domains. (<b>B</b>) 293 cells were transfected with empty vector (pcDNA) or a construct encoding CEACAM1 ΔCT-GFP (1 µg) together with increasing amounts of a plasmid encoding the HA-tagged CEACAM8/1 chimera (0 to 6 µg). One sample was transfected with CEACAM8/1-HA (6 µg) only. Differential expression of CEACAM1 ΔCT-GFP and CEACAM8/1-HA was verified by Western blotting of whole cell lysates (WCLs) using α-GFP antibody (CEACAM1 ΔCT; upper panel) or α-HA antibody (CEACAM8/1; middle panel), respectively. Equal loading of samples was demonstrated by Western blotting with α-tubulin antibody (lower panel). (<b>C, D</b>) Cells transfected as in (B) were infected with Opa_CEA-expressing gonococci. After infection for 2 h, total cell-associated bacteria (C) or recovered intracellular bacteria (D) were quantified. Numbers are expressed relative to cells expressing CEACAM1 ΔCT in the absence of CEACAM8/1. Bars represent mean ± SEM of three independent experiments done in triplicate (n = 9). Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001; *, p<0.05.</p

Membrane microdomain localization is not required for CEACAM1-initiated PI(3,4,5)P generation.

<p>(<b>A</b>) Domain organization of the used CEACAM constructs. N – Ig_V-like N-terminal domain; A1, B, A2 – Ig_C2-like domains. (<b>B</b>) 293 cells were transfected with empty vector (pcDNA) or constructs encoding HA-tagged CEACAM1, CEACAM3, or a chimeric protein consisting of the extracellular domains of CEACAM1 fused to the transmembrane domain of CEACAM3 (CEACAM1/CEACAM3-TM). Expression was verified by Western blotting with α-HA antibody. (<b>C, D</b>) Cells transfected as in (B) were pretreated for 30 min with 500 µM methyl-β-cyclodextrin (MβCD) or left untreated. After infection for 2 h with Opa_CEA-expressing gonococci total cell-associated bacteria (C) or recovered intracellular bacteria (D) were quantified. Bars represent mean ± SEM of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without MβCD treatment. Significance was tested using an unpaired, two-sided Student's t-test; **, p<0.01; n.s. – not significant. (<b>E, F</b>) Cells transfected as in (B) were pretreated with 50 µM of LY294002 (LY). Cells were infected as in (C, D) and total cell-associated bacteria (E) or recovered intracellular bacteria (F) were quantified. Bars represent mean ± SEM of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without LY treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001; **, p<0.01; n.s. – not significant. (<b>G</b>) 293 cells were transfected with mKate-tagged CEACAM1 together with GFP-tagged Btk-PH domain and treated with MβCD before infection with Pacific Blue labelled Opa_CEA-expressing gonococci. After 60 min, samples were fixed and analyzed via confocal microscopy. Arrowheads highlight gonococci associated with clustered CEACAM1. Insets show enlargement of the highlighted area. Bars represent 5 µm.</p