Article Figures & Data
Figures
- FIG 1
Genetic relationships of fnbA A domain between strains. fnbA gene segments encoding the entire FnBPA A domain were sequenced from 22 clinical isolates. Numbers indicate patient number, followed by the spa type of each strain. Sequences were aligned together with those of reference strains for the fnbA isotypes I to VII.
- FIG 2
Course of IgG levels against FnBPA isotypes following bacteremia. (A) Representative course of IgG levels against 7 FnBPA isotypes (I to VII) following the onset of bacteremia (day 0, defined as the day of the first blood culture positive for S. aureus) in patient 1, infected with a strain carrying isotype II. (B) Same plot for patient 15, infected with a strain carrying isotype V. Data points represent the mean for two separate measurements per serum sample.
- FIG 3
Variation in peak IgG levels against FnBPA isotypes after the onset of bacteremia. Scatter plot showing the distribution of peak IgG levels against all 7 FnbPA isotypes (I to VII) in 22 patients suffering from bacteremia. Each point represents one patient, and horizontal lines represent the medians and interquartile ranges.
- FIG 4
Preincubation of FnBPA isotypes with human IgG does not prevent binding of fibrinogen. (A) The most common isotypes, I to V, coupled to beads, were preincubated in this Luminex assay with a dilution range of IgG purified from polyclonal human IgG (PHG). Binding of a fixed concentration of PE-labeled fibrinogen was then measured. (B) Results of the same experiment using dilution ranges of IgG purified from different patients. Each isotype was preincubated with IgG from a patient who was infected with a strain carrying that isotype (e.g., patient 19 was infected with a strain carrying isotype I).
- FIG 5
Confirmation of the inability of human IgG to prevent binding between FnBPA isotypes and fibrinogen. (A) PE-labeled, free FnBPA isotypes II and III were preincubated with a dilution range of total IgG either from PHG or from patients 14 and 18 (who were infected with strains carrying isotypes II and III, respectively). Protein-antibody complexes were then allowed to bind fibrinogen coupled to Luminex beads. (B) Using an alternative ELISA, preincubation of fibrinogen with IgG from patient 15 (infected with a strain carrying isotype V) did not clearly affect subsequent binding to coated FnBPA isotype V. Similar results were obtained for isotype I (data not shown). (C) Using the same ELISA, preincubation with ClfA, instead of IgG, inversely decreased the amount of remaining fibrinogen bound to FnBPA isotype V. All data points represent the mean for two measurements and are expressed as median fluorescence intensity (MFI) for panel A or as optical density at 450 nm for panels B and C.
Supplemental Material
TABLE S1
Initial-to-peak fold increases in specific IgG levels for FnBPA isotypes in bacteremia patients. A fold increase of 1.0 means that the IgG level did not change during the course of infection compared to that of the initial measurement at the onset of bacteremia, whereas a fold increase of, e.g., 23.1 means that the peak IgG level in that patient after onset of infection was 23.1 times higher than the initial measurement. The FnBPA isotype of the infecting S. aureus strain, as determined with sequencing, is indicated in the second column. Significant initial-to-peak increases in IgG of more than 4-fold are indicated in italic boldface. Download TABLE S1, DOCX file, 0.01 MB.
Copyright © 2018 den Reijer et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
Validation of fibrinogen binding by FnBPA isotypes. (A) Increasing concentrations of PE-labeled fibrinogen bind to all FnBPA isotypes but not to the control proteins (IsdA, SdrE, and ClfB) in a dose-dependent and saturable manner. (B) Adding increasing concentrations of unlabeled fibrinogen to a fixed amount of 1.5 μg of PE-labeled fibrinogen shows competition for the binding to all FnBPA isotypes. Data points represent the means for two separate measurements. Download FIG S1, TIF file, 0.4 MB.
Copyright © 2018 den Reijer et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Specific and dose-dependent binding of human IgG to FnBPA isotypes I to V. (A) Beads coated with recombinant FnBPA isotypes I to V were incubated in a Luminex setup with a dilution range of total IgG purified from polyclonal human IgG (PHG) or patients (p14 to p19; for each FnBPA isotype, serum was chosen from a patient who was infected with a strain carrying that isotype). (B) FnBPA isotypes applied as a coating onto wells (0.5 µg/well) were incubated in an alternative ELISA setup with a dilution range of the same IgG, purified from PHG or patients. A plateau in antibody binding at higher concentrations can be observed, indicating that only FnBPA-specific IgG within the polyclonal antibodies binds. Download FIG S2, TIF file, 0.4 MB.
Copyright © 2018 den Reijer et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
