Functional blocking of Staphylococcus aureus adhesins following growth in ex vivo media

Abstract

Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteria grown in culture media that more closely reflect the human milieu than conventional broth. This study examined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing a complex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. The adherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite the expresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in the acquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to contain fibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialysate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on the culture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solid surfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound to protein A following contact with host proteins. These findings have important implications for future studies of S. aureus adhesins.

FIG. 1.

Functional blocking of S. aureus adhesins following growth in dialysate. The adherence of S. aureus strains to fibronectin (a) and fibrinogen (b) was assessed by microtiter plate assay following growth in dialysate and THB. The data are shown as the mean ± standard error of the mean. OD₄₀₅, optical density at 405 nm.

FIG. 2.

Surface expression of S. aureus adhesins. Western immunoblots demonstrate the cell wall-associated proteins ClfA, ClfB (strain Newman), and FnBPA (strain 8325-4) extracted from bacteria following growth in dialysate or THB.

FIG. 3.

Fibronectin, fibrinogen, and immunoglobulins are present in dialysate and become associated with the surface of S. aureus. Western immunoblotting was used to detect the presence of human proteins in dialysate concentrated 20-fold and in the surface-associated layer acquired by S. aureus following growth in dialysate. Purified human proteins were used as positive controls. Lanes 1, 2, and 3, purified fibronectin, dialysate, and surface coat, respectively, probed with anti-fibronectin antibodies; lanes 4, 5, and 6, purified fibrinogen, dialysate, and surface coat, respectively, probed with anti-fibrinogen antibodies; lanes 7, 8, and 9, purified immunoglobulins (classes IgG, IgM, and IgA), dialysate, and surface coat, respectively, probed with antibodies to IgG, IgM, and IgA.

FIG. 4.

S. aureus grown in dialysate acquires a surface coat. Transmission electron microscopy of S. aureus strain Newman cultured overnight in THB (a) and dialysate (b). The bacteria acquire an electron-dense surface layer during growth in dialysate. Magnification, ×48,000.

FIG. 5.

Expression of protein A and its interaction with immunoglobulins interfere with bacterial adherence to fibrinogen following growth in dialysate. Shown are adherence of wild-type (WT) S. aureus strains Newman, V8, and Wood; Newman and V8 mutants defective in protein A (spa−); and Wood complemented with a multicopy plasmid expressing protein A (spa+) following growth in dialysate (wild-type Wood is naturally deficient in protein A). Also shown is the adherence of wild-type Newman, V8, and Wood following growth in IgG-depleted dialysate. The data are shown as the mean ± standard error of the mean. OD₄₀₅, optical density at 405 nm.