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