The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis

Abstract

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.

Fig. 1.

(a) Flow cytometry histogram showing the fluorescence of S. aureus strain Reynolds (CP5) and its isogenic capsule-negative mutant (CP⁻) after transformation with pCM29-GFP plasmid. (b) Flow cytometry histogram showing binding of rabbit anti-CP5 antibodies to GFP-labelled Reynolds (CP5) and isogenic mutant (CP⁻), detected with Alexa647-conjugated protein A. (c) Transmission electron micrographs showing the GFP-labelled Reynolds (CP5) and mutant (CP⁻) strain. Strains were pretreated with anti-CP5 antibodies to enhance stability and electron density of the capsule. Representative images are shown.

Fig. 2.

Phagocytosis of GFP-labelled Reynolds (CP⁻ and CP5) by purified human neutrophils in the presence of human plasma alone (a) or plasma with 0.5 µM Efb (b, c), measured by fluorescence (geomean) of the neutrophils. (a) The polysaccharide capsule of S. aureus provides protection against phagocytosis at low plasma concentrations. (b) Addition of exogenous Efb inhibits phagocytic uptake of unencapsulated strain Reynolds (CP⁻) at all tested plasma concentrations. (c) Inhibition of phagocytosis of encapsulated strain Reynolds (CP5) is enhanced by addition of Efb. Graphs represent mean ± sd of three separate experiments. *P<0.05, **P<0.005 for Reynolds (CP5) versus Reynolds (CP⁻) or Efb versus buffer (two-tailed Student’s t-test). MFI, mean fluorescence intensity.

Fig. 3.

(a) Phagocytosis of different GFP-labelled CP⁻-, CP5- and CP8-expressing S. aureus strains in the presence of 1, 3 or 10 % human plasma. Phagocytosis is displayed as the relative fluorescence compared with the 10 % plasma condition of each strain. Graph represents mean ± sd of three separate experiments. *P<0.05, **P<0.005 for 1 or 3 % plasma versus 10 % plasma of the same strain (two-tailed Student’s t-test). (b) Addition of Efb inhibits phagocytic uptake of different CP5 and CP8 encapsulated S. aureus strains at both 1 and 10 % human plasma. This was displayed by the relative fluorescence (geomean; compared with the buffer condition at 10 % plasma of the same strain) of the neutrophils. Graph represents mean ± sd of three separate experiments. *P<0.05, **P<0.005 for Efb versus buffer of the same strain (two-tailed Student’s t-test). MFI, mean fluorescence intensity.

Fig. 4.

Interplay between Efb and capsule on other isogenic mutants of CP5 and CP8. (a) Binding of rabbit CP5 and CP8 antibodies to different FITC-labelled CP5- and CP8-expressing S. aureus strains. (b–e) Phagocytosis of different FITC-labelled CP5- and CP8-expressing S. aureus strains by purified neutrophils in the presence of 1 or 10 % human plasma and 0.5 µM Efb. (b) Phagocytosis of strain Reynolds (CP5) and Reynolds (CP⁻). (c) Phagocytosis of CP5-expressing strain Newman and its isogenic mutant Newman (CP⁻) (buffer vs Efb: P<0.05 at 1 and 10 % plasma). (d) Phagocytosis of CP8-expressing strain Becker and its isogenic mutant Becker (CP⁻) (buffer vs Efb: n.s. at 1 % plasma, P<0.05 at 10 % plasma). (e) Phagocytosis of CP8-expressing strain MN8 and its isogenic mutant MN8 (CP⁻) (buffer vs Efb: P<0.05 at 1 and 10 % plasma). Graphs represent mean ± sd of three separate experiments. At 10 % plasma, the inhibitory effect of Efb was statiscally significant for all strains, but at 1 % plasma was significant only for strains Newman and MN8. MFI, mean fluorescence intensity.

Fig. 5.

(a) Confocal images of the binding of Alexa488-labelled fibrinogen to mCherry-labeled Reynolds (CP⁻ and CP5) strains, pre-opsonized with human serum (3 %), in the presence of Efb variants (0.5 µM). Representative images are shown. (b) Flow cytometry analyses of samples shown in (a). Graph represents mean ± sd of three separate experiments. **P<0.005 for Efb versus buffer, EfbΔFg or EfbΔC3 (two-tailed Student’s t-test). MFI, mean fluorescence intensity.