Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , The relevance of surface bound vitronectin was analyzed by challenging intact bacteria with complement active human serum in which vitronectin was depleted (HSΔ Vn ). P . aeruginosa strain SG137 was incubated in HSΔ Vn diluted in GVB++ buffer. After incubation, the cells were plated on NB agar plates and the number (CFU) of surviving bacteria was determined. B , The relevance of clusterin was analyzed by challenging intact bacteria with complement active human serum in which clusterin activity was blocked. Bacteria were incubated with anti-clusterin mAb or mouse IgG and were thereafter challenged with NHS (1%) diluted in GVB++ buffer. Incubation of bacteria in 1% HiNHS was used as a negative control. Number of bacteria (CFU) at the initiation of all experiments was defined as 100%. The mean values from three independent experiments are shown with error bars indicating SD. *, p ≤ 0.05;**, p ≤ 0.01.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: Incubation, Activity Assay, Negative Control

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Binding of clusterin to P . aeruginosa strain SG137 was assayed by flow cytometry. Bacteria were incubated with clusterin (5–25 μg/ml) and bound clusterin was detected with a monoclonal clusterin antibody and Alexa488-labeled mouse antiserum. Bacteria incubated with a monoclonal clusterin antibody and Alexa488-labeled mouse antiserum served as controls. B , Clusterin binds to intact P . aeruginosa and binding was dose-dependent. Four laboratory strains of P . aeruginosa were analyzed for clusterin binding using a whole cell ELISA. Whole bacteria were immobilized onto microtiter plates and clusterin (1–5 μg/ml) was added. Bound clusterin was detected with a monoclonal clusterin antibody followed by HRP-conjugated anti-mouse. C , Clusterin bind to P . aeruginosa . P . aeruginosa strains SG137, ATCC 27853, NCTC 10662 and PAO1 when incubated with NHS. Bacteria were washed, lysed, separated by SDS-PAGE and analysed by Western blotting. Bound clusterin was detected with a monoclonal clusterin antibody and HRP-conjugated anti-rabbit. A representative experiment of three is shown. D , Clusterin bound to both laboratory and clinical P . aeruginosa strains. Binding of clusterin (2.5 μg/ml) to immobilized bacteria (0.5x10 7 ) was assayed by ELISA. Bound clusterin was detected with a monoclonal clusterin antibody followed by HRP-conjugated anti-rabbit pAb. E , Binding of vitronectin and clusterin from NHS to immobilized P . aeruginosa were tested by whole cell ELISA. Bound vitronectin or clusterin was detected with the polyclonal vitronectin antiserum or monoclonal clusterin antibody followed by HRP-conjugated anti-rabbit or mouse pAb. The mean values of three independent experiments and standard deviations (SD) are presented. Statistical significance of differences was estimated using Student’s t test. *, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: Binding Assay, Flow Cytometry, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Lpd and BSA were separated by SDS-PAGE and the two proteins were identified by silver staining. The position of the marker proteins is presented on the left. B, Vitronectin binds to Lpd. Lpd and BSA were transferred to a membrane and the membrane was incubated with vitronectin (20 μg/ml). Bound vitronectin was detected with vitronectin specific antiserum and HRP-conjugated anti-rabbit. BSA was used as a negative control. C , Vitronectin bound to immobilized Lpd and binding was dose-dependent. Binding of vitronectin (0.01–12.5 μg/ml) to immobilized Lpd was assayed by ELISA. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. D , Vitronectin binds to Lpd with an affinity of 600 ± 50 nM. Binding of Lpd or BSA used at various concentrations (0.003–50 μM) to NT-647-labeled vitronectin (50 nM) was evaluated in fluid phase by microscale themophoresis (MST). Thermophoresis was recorded at 50% LED power and 80% MST power for 30 s in a Monolith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment was plotted against the concentration of Lpd. E , Clusterin binds to Lpd. Lpd and BSA were separated by SDS-PAGE, transferred to a membrane and incubated with vitronectin (20 μg/ml) Bound clusterin was detected with a monoclonal clusterin antibody and HRP-conjugated anti-mouse. BSA was used as a negative control. F , Clusterin bound to immobilized Lpd and binding was dose-dependent. Binding of clusterin (0.01–10 μg/ml) to immobilized Lpd was assayed by ELISA. Bound clusterin was detected with anti-clusterin mAb and HRP-conjugated anti-mouse pAb. G , Clusterin binds to Lpd with an affinity of 654 ± 54 nM. Binding of Lpd or BSA used at various concentrations (0.001–50 μM) to NT-647-labeled clusterin (25 nM) was evaluated in fluid phase by microscale themophoresis. Thermophoresis was recorded at 50% LED power and 80% MST power for 30 s in a Monolith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment was plotted against the concentration of Lpd.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: SDS Page, Silver Staining, Marker, Incubation, Negative Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Fluorescence, Concentration Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Localization of the region within vitronectin that mediates binding to Lpd. Vitronectin uses one contact region i.e. aa 354–363 to contact Lpd. Vitronectin 80–396 (Vn 80-396 ) and seven deletion mutants were expressed in HEK cells and purified. The numbers refer to amino acids residues that are included in each construct ( left panel ). Black indicates the heparin binding regions of Vitronectin ( left panel ). Binding of serum purified full-length vitronectin and vitronectin deletion mutants (5 μg/ml) to immobilized Lpd was assayed by ELISA ( right panel ). Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. B , Heparin inhibits binding of vitronectin to Lpd and the effect was dose-dependent. The effect of heparin (0.01–5 mg/ml) on vitronectin binding to immobilized Lpd was assayed. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. C , Schematic picture of full length Lpd and its fragments. D , The vitronectin-binding regions are located within two separate binding domains of Lpd. Equimolar amounts (13.3 nM) of full length Lpd and Lpd deletion mutants were immobilized onto microtiter plates, vitronectin was added and bound vitronectin was quantified. E , The clusterin-binding regions are located within two separate binding domains of Lpd. Equimolar amounts (13.3 nM) of full length Lpd and Lpd deletion mutants were immobilized onto microtiter plates, clusterin was added and bound clusterin was quantified. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: Binding Assay, Purification, Construct, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A, Effect of increasing clusterin levels in the presence of a constant concentration of vitronectin. Binding of clusterin (used at the indicated concentrations) and vitronectin (5 μg/ml) to immobilized Lpd was analysed by ELISA. Bound clusterin was detected with anti-clusterin mAb (■) and bound vitronectin was detected with anti-vitronectin mAb (◇). B, In a reverse setting, the clusterin concentration was kept constant (2.5 μg/ml) and binding of vitronectin (used at the indicated concentrations) was evaluated. The mean values of three independent experiments and SD are presented.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: Both vitronectin ( A ) and clusterin ( B ) when bound to immobilized Lpd inhibits C5b-9 deposition. A , Vitronectin (10–50 μg/ml) or Factor H (10–50 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. B , Clusterin (2.5–20 μg/ml) or Factor H (2.5–20 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. ***, p ≤ 0.001.

Article Snippet: Monoclonal mouse anti-human clusterin was purchased from TecoMedical (Bünde, Germany) and monoclonal mouse anti-human vitronectin 58–1 from Abcam (Cambridge, UK).

Techniques: Incubation