Staphylococcal Protein A Is a Key Factor in Neutrophil Extracellular Traps Formation

Abstract

Staphylococcus aureus are strong inducers of neutrophil extracellular traps (NETs), a defense mechanism of neutrophils against pathogens. Our aim was to explore the role of Protein A in S. aureus-induced NETosis. We determined the Protein A production of four different S. aureus strains and found a direct relationship between the degree of NETosis induction and Protein A production: strains producing higher concentrations of Protein A evoke significantly more NETs. A S. aureus strain in which Protein A as well as a second binding protein for immunoglobulins (Sbi) have been knocked-out (ΔSpA ΔSbi) induced significantly less NETosis than the wild-type strain. NETosis induction by this knockout strain can be rescued by the addition of purified Protein A. Dead S. aureus did not induce NETosis. In conclusion, Protein A is a determinant for NETosis induction by S. aureus.

Keywords: NETs; Protein A; S. aureus; SpA; Staphylococcus aureus; neutrophil extracellular traps.

Figure 1

Protein A production by Staphylococcus aureus. (A) Protein A secretion is higher in S. aureus strains Newman and USA300 compared to S. aureus strains M116 and RN6390, as determined by an ELISA assay (n = 5). (B) More Protein A is bound to the surface of S. aureus strains Newman and USA300 compared to S. aureus strains M116 and RN6390, as determined by FACS (n = 3). (C) Amount of Protein A bound to the surface of S. aureus measured on 2.5, 3.5, and 24 h as determined by FACS. Except for strain RN6390, the amount of surface bound Protein A is increasing over time (n = 3, neutrophils derived from three individual donors).

Figure 2

Neutrophil extracellular traps (NETs) induction by different Staphylococcus aureus strains Newman, USA300, M116, and RN6390. (A) In vitro NETs formation as indicated by propidium iodide (red). (B) Strains Newman and USA300 induce significantly more NETs than M116 and RN6390, as indicated by percentage of NETs coverage in the total volume. Results of three separate experiments, neutrophils were derived from three individual donors (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3

Reversed effect of significantly reduced NETosis by Staphylococcus aureus knockout strain after the addition of purified Protein A. Except for S. aureus Newman ΔSpAΔSbi, all conditions induce more NETosis than Protein A only. Results of three separate experiments, neutrophils were derived from three individual donors. * indicates significant difference from S. aureus Newman ΔSpAΔSbi. ^# indicates significant difference when compared to neutrophils stimulated with 0.9 µg/ml Protein A (*/^#p < 0.05; **/^##p < 0.01; ***/^###p < 0.001).

Figure 4

The effect of live and dead Staphylococcus aureus and the presence of Protein A (both produced by S. aureus and added) on NETosis in different rescue experiments.

Figure 5

The effect of Protein A on dead bacteria in the presence of neutrophils after 40 min of incubation. (A) Dead Staphylococcus aureus Newman incubated with neutrophils. Arrows indicate phagocytosis. (B) Dead S. aureus Newman incubated with 0.9 µg/ml Protein A and neutrophils. Blue, DNA; red, dead bacteria.