Figure 5.
Figure 5. Antibody against the repeat domain of coagulase promotes OPK of staphylococci. (A) Anticoagulated human plasma or serum were inoculated with 5 × 106 CFU S. aureus Newman (WT), Δcoa/Δvwb, or MRSA isolate USA300 LAC and incubated for 60 min before dilution and plating for CFU. Agglutinated staphylococci were released by SK treatment. (B and C) Anticoagulated blood from human volunteers was inoculated with 5 × 106 CFU S. aureus USA300 (B) or 5 × 106 CFU S. epidermidis (C), incubated for 60 min, and CFU enumerated with or without SK treatment. Blood samples were pretreated with CD to block phagocytosis. (D) Addition of mAb 3B3 to blood samples promoted OPK of S. aureus USA300. Data were collected as in B. (E) Anticoagulated mouse blood with or without mAb 3B3 was inoculated with S. aureus Newman (pGFP), S. aureus coaΔR (pGFP), or left uninfected. At 0, 30, and 60 min, extracellular bacteria were first killed with lysostaphin and neutrophils were stained with α-GR1. The mean fluorescence intensity (MFI) of GFP was used as a measure for phagocytosed bacteria. (F) Mouse blood was supplemented with 5% Alexa Fluor 488–conjugated human fibrinogen. Incorporation of Alexa Fluor 488-fibrinogen into fibrin and association with neutrophils was measured by FITC fluorescence. (G) Mouse blood was incubated for 30 min with wild-type S. aureus in the absence or presence of mAb 3B3, stained with Giemsa, and viewed by microscopy. Bar, 10 µm. (H) S. aureus Newman was incubated with anticoagulated mouse blood without or with CD and without or with mAb 3B3; staphylococcal survival and replication was assessed by CFU enumeration at timed intervals. (I) mAb 3B3 or IgG1 isotype control were administered into the peritoneal cavity of mice (n = 10 per experiment). Animals were challenged by intravenous injection with S. aureus Newman or the coaΔR variant. After 30 min, animals were bled via cardiac puncture and CFU enumerated. (A-D) Experiments were performed in duplicate, results averaged, and data were recorded as percent inoculum. (E, F, H, and I) Data are representative of two independent analyses conducted in triplicate. Error bars indicate SEM. Statistical analyses were performed with the two-tailed Student’s t test (A–D and J) or with two-way ANOVA with Bonferroni post-test (E, F, and H). *, P < 0.05 and **, P < 0.01.

Antibody against the repeat domain of coagulase promotes OPK of staphylococci. (A) Anticoagulated human plasma or serum were inoculated with 5 × 106 CFU S. aureus Newman (WT), Δcoa/Δvwb, or MRSA isolate USA300 LAC and incubated for 60 min before dilution and plating for CFU. Agglutinated staphylococci were released by SK treatment. (B and C) Anticoagulated blood from human volunteers was inoculated with 5 × 106 CFU S. aureus USA300 (B) or 5 × 106 CFU S. epidermidis (C), incubated for 60 min, and CFU enumerated with or without SK treatment. Blood samples were pretreated with CD to block phagocytosis. (D) Addition of mAb 3B3 to blood samples promoted OPK of S. aureus USA300. Data were collected as in B. (E) Anticoagulated mouse blood with or without mAb 3B3 was inoculated with S. aureus Newman (pGFP), S. aureus coaΔR (pGFP), or left uninfected. At 0, 30, and 60 min, extracellular bacteria were first killed with lysostaphin and neutrophils were stained with α-GR1. The mean fluorescence intensity (MFI) of GFP was used as a measure for phagocytosed bacteria. (F) Mouse blood was supplemented with 5% Alexa Fluor 488–conjugated human fibrinogen. Incorporation of Alexa Fluor 488-fibrinogen into fibrin and association with neutrophils was measured by FITC fluorescence. (G) Mouse blood was incubated for 30 min with wild-type S. aureus in the absence or presence of mAb 3B3, stained with Giemsa, and viewed by microscopy. Bar, 10 µm. (H) S. aureus Newman was incubated with anticoagulated mouse blood without or with CD and without or with mAb 3B3; staphylococcal survival and replication was assessed by CFU enumeration at timed intervals. (I) mAb 3B3 or IgG1 isotype control were administered into the peritoneal cavity of mice (n = 10 per experiment). Animals were challenged by intravenous injection with S. aureus Newman or the coaΔR variant. After 30 min, animals were bled via cardiac puncture and CFU enumerated. (A-D) Experiments were performed in duplicate, results averaged, and data were recorded as percent inoculum. (E, F, H, and I) Data are representative of two independent analyses conducted in triplicate. Error bars indicate SEM. Statistical analyses were performed with the two-tailed Student’s t test (A–D and J) or with two-way ANOVA with Bonferroni post-test (E, F, and H). *, P < 0.05 and **, P < 0.01.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal