Article Figures & Data
Figures
- FIG 1
sdrD promoter activity and SdrD protein expression under different growth conditions. (A) Promoter activity of sdrD during growth in TSB using NCTC8325-4 harboring the sdrD-GFP reporter construct (S. aureus subsp. aureus psdrD-GFP). Data represent the means ± SEMs from an individual experiment. The experiments were performed twice in triplicate. RFU, relative fluorescence units. (B) Immunoblotting of the bacterial lysates and the culture cell-free supernatant of NCTC8325-4 and its isogenic mutant, NCTC8325-4 ΔsdrD, using anti-SdrD antibody on the bacterial lysates and the culture cell-free supernatant. A representative Western blot is shown. (C) Promoter activity of sdrD evaluated by fluorescence microscopy using NCTC8325-4 harboring psdrD-GFP in the presence of freshly isolated human neutrophils. (Top) Live imaging was performed after 0.2, 1.5, and 3.5 h using fluorescence microscopy; (bottom) bright-field and GFP merged images. The white boxes in the first column labeled 3.5 h are enlarged in the second column labeled 3.5 h. The experiment was performed at an MOI of 20. Arrows, S. aureus subsp. aureus psdrD-GFP. The sizes of the scale bars are indicated.
- FIG 2
SdrD promotes S. aureus survival in human and mouse whole blood. (A and B) SdrD increases the rate of survival of NCTC8325-4 in human blood (A) and in mouse blood (B). The rate of survival for bacteria inoculated at time zero was arbitrarily set equal to 100%, and the rate of survival after 3 h is presented as a percentage of the number of bacteria in the inoculum. (C) Transcomplementation of NCTC8325-4 ΔsdrD with plasmid-expressed SdrD (left) and ectopic expression of SdrD in L. lactis (right) restore survival in human whole blood. Bacterial survival was analyzed by viability counting. The rate of survival is presented as described in the legend to panel A. (D) Influence of rSdrD (12 μg/ml) on the survival of NCTC8325-4 and NCTC8325-4 ΔsdrD in human whole blood. The rate of survival in untreated blood was arbitrarily set equal to 1, and the rate of bacterial survival in blood treated with rSdrD is represented as the fold change. (E) The survival of E. coli and S. haemolyticus in human whole blood is increased in the presence of rSdrD (12 μg/ml). The fold change in bacterial survival is presented as described in the legend to panel D. (F) NCTC8325-4 and its mutant, NCTC8325-4 ΔsdrD, were labeled with FITC, and 40 μl of different concentrations of inoculum (∼1 × 108 or 1 × 107 CFU/ml) was incubated with human whole blood. Data represent the geometric mean of the fluorescence intensity (GMFI). Data are presented as the means ± SEMs from at least three independent experiments. Statistical analysis was performed by Student's t test. Significant differences are indicated by asterisks. *, P < 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.
- FIG 3
SdrD promotes S. aureus survival in the presence of human neutrophils. (A and B) Survival of NCTC8325-4 and NCTC8325-4 ΔsdrD (A) and L. lactis containing sdrD-pMG36e or the empty vector (B) after incubation with freshly isolated human neutrophils. The bacteria were opsonized with plasma and exposed to purified neutrophils before bacterial survival was analyzed by viability counting. The rate of survival for the bacterial inoculum was arbitrarily set equal to 100%, and the rate of survival of the bacteria after 1 h is presented as a percentage of the number of bacteria in the inoculum. (C) Influence of rSdrD on survival of plasma-opsonized NCTC8325-4 or NCTC48325-4 ΔsdrD in the presence of human neutrophils. Bacterial survival was analyzed by viability counting. The survival of untreated neutrophils was arbitrarily set equal to 1, and the rate of bacterial survival in the neutrophils treated with rSdrD is presented as the fold change. (D) PMA-induced neutrophils were used to perform a NET-mediated killing assay using NCTC8325-4 or NCTC48325-4 ΔsdrD at an MOI of 1. The bacteria that were incubated without neutrophils served as controls, and the rate of survival for these bacteria was arbitrarily set equal to 100%. The rate of survival of these bacteria after 30 min is presented as a percentage of the number of bacteria in the inoculum. (E) Freshly isolated neutrophils were exposed to NCTC8325-4 or NCTC48325-4 ΔsdrD at an MOI of either 10 or 20. Percent viability was calculated by measuring the amount of LDH released from the cytosol of damaged cells into the supernatant. Data represents means ± SEMs from at least three independent experiments. Statistical analysis was performed by Student's t test. Significant differences are indicated by asterisks. *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 4
SdrD increases the bacterial burden in a murine systemic infection model. (A) The bacterial loads in the kidney, liver, spleen, and blood (number of CFU per gram for organs and number of CFU per milliliter for blood) of 8-week-old female CD-1 mice were enumerated 4 h and 4 days after systemic infection with NCTC8325-4 and NCTC8325-4 ΔsdrD. The data represent the means ± SEMs from an individual experiment for data obtained at 4 h postinfection and two independent experiments for data obtained at 4 days postinfection. Statistical analysis was performed by Student's t test. Significant differences are indicated by asterisks. *, P < 0.05; **, P ≤ 0.01. (B) Thin sections of H&E-stained uninfected kidney tissue and kidney tissue infected with S. aureus subsp. aureus NCTC8325-4 and NCTC8325-4 ΔsdrD collected on day 4 postinfection were analyzed by light microscopy. Arrows, infiltrate of neutrophils. The sizes of the scale bars are indicated.
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1. S. aureus growth under different experimental conditions.
PDF, 123K
- Supplemental file 2 -
Fig. S2. SdrD does not affect whole-blood phagocytosis.
PDF, 75K
- Supplemental file 3 -
Fig. S1 and S2 legends.
PDF, 77K
- Supplemental file 1 -
