Journal: Journal of Bacteriology

Article Title: The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis

doi: 10.1128/JB.00184-21

Figure Lengend Snippet: SplB inhibits the alternative complement pathway. To probe the spontaneous activation of the alternative pathway, microtiter plates were coated with LPS. Increasing concentrations of SplB or BSA were incubated with appropriately diluted NHS as the source of complement for 1 h, and the mixtures were added to the microtiter plates. After incubation for 20 min at 37°C, the plates were washed, and deposited C3b or C5b-9 was determined with specific antisera or monoclonal antibodies. SplB but not BSA diminished the deposition of C3b (A) and C5b-9 (B).

Article Snippet: Human plasma-purified complement proteins C1q, C2, C3, C3b, C4, C4b, C5, C5b, C6, C7, C8, and C9; factor H; and C4BP were purchased from CompTech (Complement Technology Inc.) (Tyler, TX, USA).

Techniques: Activation Assay, Incubation, Bioprocessing

Journal: Journal of Bacteriology

Article Title: The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis

doi: 10.1128/JB.00184-21

Figure Lengend Snippet: SplB inhibits the lectin and classical complement pathways. The effects of SplB on complement activation and the deposition of C3b and C5b-9 were analyzed as described in the legend to . (A and B) The lectin pathway was activated via mannan-coated microtiter plates. SplB interfered with the lectin pathway in a concentration-dependent manner, lowering the deposition of C3b (A) and C5b-9 (B). (C and D) To test for interference with the classical pathway, the microtiter wells were coated with IgM. Again, SplB reduced the classical pathway-mediated deposition of C3b (C) and C5b-9 (D) concentration dependently.

Article Snippet: Human plasma-purified complement proteins C1q, C2, C3, C3b, C4, C4b, C5, C5b, C6, C7, C8, and C9; factor H; and C4BP were purchased from CompTech (Complement Technology Inc.) (Tyler, TX, USA).

Techniques: Activation Assay, Concentration Assay

Journal: Journal of Bacteriology

Article Title: The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis

doi: 10.1128/JB.00184-21

Figure Lengend Snippet: SplB and aureolysin reduce the deposition of C3b and C5b-9 on the surface of S. aureus cells. (A) SplB or BSA (1 μM) was incubated with complement-competent NHS (5%) for 1 h, and the mixture was added to spa -deficient S. aureus cells (RN1HF Δ spa ) for 20 min at 37°C. C3b or C5b-9 on the bacterial cell surface was stained with specific fluorescent antibodies and evaluated by microscopy. In the control samples (BSA), there was abundant deposition of C3b (I, top, green) and C5b-9 (II, top, red) on the bacterial surfaces. SplB strongly interfered with complement deposition (C3b [I, bottom, green] and C5b-9 [II, bottom, red]). Panels III show bacterial DNA stained with 4′,6-diamidino-2-phenylindole (DAPI), and panels IV show the overlays of the three images. Bars, 100 μm. (B and C) SplB, aureolysin, or both were incubated with complement-active NHS (5%) for 1 h at the indicated concentrations. SplC and BSA served as controls. The resulting solutions were added to spa -deficient S. aureus cells (RN1HF Δ spa ) for 20 min at 37°C. After washing, the deposition of C3b (B) or C5b-9 (C) was stained with specific antibodies and fluorescent secondary reagents and evaluated by flow cytometry. SplB and aureolysin inhibited the deposition of C3b and C5b-9 on the bacterial surface in a concentration-dependent manner. The combination of SplB and aureolysin had the strongest effect, completely abolishing the deposition of C3b (B) and reducing the deposition of C5b-9 by 90% (C). SplC and BSA, the negative controls, had no influence on complement deposition (B and C). Depicted are means ± SD from three independent experiments.

Article Snippet: Human plasma-purified complement proteins C1q, C2, C3, C3b, C4, C4b, C5, C5b, C6, C7, C8, and C9; factor H; and C4BP were purchased from CompTech (Complement Technology Inc.) (Tyler, TX, USA).

Techniques: Incubation, Staining, Microscopy, Control, Flow Cytometry, Concentration Assay

Journal: PLoS ONE

Article Title: Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

doi: 10.1371/journal.pone.0040489

Figure Lengend Snippet: Sheep erythrocytes opsonized with antibodies were incubated with 0.125% NHS to visualize the activity of the classical pathway of the human complement system. Serum was pre-incubated for 10 min at 37°C with various concentrations (2.5–60 µg/ml, i.e. 54–1300 nM) of SMSB3 and its four mutants ( A ), SMSB4 and its two mutants (0.1–5 µg/ml, i.e. 2–93 nM) ( B ) and BSA as a negative control. After 1 h of incubation of NHS with erythrocytes at 37°C, the degree of complement-mediated lysis was estimated by measurement of released hemoglobin. The lysis obtained in the absence of SMSs was defined as 100% hemolytic activity. An average of three independent experiments performed in duplicate is presented with bars indicating SEM.

Article Snippet: A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8).

Techniques: Incubation, Activity Assay, Negative Control, Lysis

Journal: PLoS ONE

Article Title: Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

doi: 10.1371/journal.pone.0040489

Figure Lengend Snippet: Complement deposition assays were performed on microtiter plates coated with aggregated IgG (classical pathway), mannan (lectin pathway) and zymosan (alternative pathway) in presence or absence of scabies mite serpins, respectively. Shown are means ± SEM of n = 4 independent experiments, each performed in duplicate. The three pathways were measured using 1% (CP), 2% (LP) and 4% (AP) NHS. Assays were performed at inhibitor protein concentrations of 25–400 µg/ml (0.5–8.6 µM) for SMSB3/wt and SMSB3/B and 10–200 µg/ml (0.2–3.7 µM) for SMSB4/wt. Significant differences between wild type SMSB3 and the hinge mutant SMSB3/B (*; p<0.05).

Article Snippet: A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

doi: 10.1371/journal.pone.0040489

Figure Lengend Snippet: Microtiter plates were coated with various purified human complement proteins or BSA as a negative control and incubated with 20 µg/ml SMSB3 ( A ) or SMSB4 ( B ). Bound serpins were detected using specific polyclonal antibodies against SMSB3 and SMSB4. Shown are means ± SEM of n = 3 independent experiments. The statistical significance of differences between BSA and the rest of the data groups was estimated using one-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001. Grey, serine proteases; black, other complement factors. C Direct binding of complement factors from NHS to SMSs. Increasing concentrations of NHS were added to wells coated with SMSB3 (▾, black), SMSB4 (•, grey) or BSA as a negative control (○, white) and bound complement factors were detected by specific antibodies. Shown are means ± SEM of n = 3 independent experiments measured in duplicates. NHS was tested from 0–100%. Positive controls were used for complement proteins, where no binding to the SMSs was detectable confirming strong immunodetection of the complement factor on 1% NHS coating.

Article Snippet: A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8).

Techniques: Purification, Negative Control, Incubation, Binding Assay, Immunodetection

Journal: Journal of Biological Chemistry

Article Title: Targeting of Functional Antibody-Decay-accelerating Factor Fusion Proteins to a Cell Surface

doi: 10.1074/jbc.m100436200

Figure Lengend Snippet: FIG. 6. Inhibition of cell surface C3 deposition by IgG-DAF fusion proteins. Control unlabeled or dansyl-labeled CHO cells were incubated with 100 nM CH1-DAF or CH3-DAF (15 min/37 °C) and then sensitized to complement with anti-CHO cell membrane antiserum. Human serum depleted of C8 was then added to a final concentration of 10%. Following incubation (45 min, 37°C), cells were washed, and C3 deposition was measured by flow cytometry using FITC-conjugated anti-C3 antibody. Histograms show relative fluorescence, with mean relative fluorescence indicated next to each peak. The data are repre- sentative of four experiments.

Article Snippet: The cells were then sensitized with anti-CHO cell membrane antiserum (10%), and NHS depleted of complement protein C8 (Quidel, San Diego, CA) added to a final concentration of 10%.

Techniques: Inhibition, Control, Labeling, Incubation, Membrane, Concentration Assay, Flow Cytometry, Fluorescence