Expression of the biofilm-associated protein interferes with host protein receptors of Staphylococcus aureus and alters the infective process

Abstract

The adherence of Staphylococcus aureus to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections. S. aureus presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on S. aureus adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.

FIG. 1.

Adhesion of S. aureus V329 and m556 to immobilized fibrinogen (A) and fibronectin (B). Wells in ELISA plates were coated with different concentrations of human fibrinogen or fibronectin. Adherence of strain V329 and the Bap mutant m556 was indicated by staining with crystal violet and measuring the absorbance at 570 nm. Data are the average results ± standard deviations of triplicate determinations. Background values of bacteria adhering to BSA-coated wells were subtracted.

FIG. 2.

Adhesion of Bap-positive S. aureus Newman and P1 strains to immobilized fibrinogen (A) and fibronectin (B). Data are the average results ± standard deviations of triplicate determinations of the increase of bacterial adherence to uncoated versus protein-coated surfaces. (C) Biofilm formation of the complemented S. aureus Newman and P1 strains. ∗, P < 0.001 (t tests).

FIG. 3.

Visualization of ClfA protein (A) and FnBPs (B) by Western blotting and ligand affinity blots, respectively. Positions of protein size markers are shown at the left of each panel. Bands corresponding to native and truncated ClfA proteins, FnBPs, and Spa are identified. Similar amounts of ClfA, FnBPs, and protein A released into the supernatant by protoplasts stabilized in raffinose were observed in V329 and m556.

FIG. 4.

Ultrathin sections of Bap-positive and Bap-negative strains. Note the presence of extracellular polysaccharide material (arrow) stained with alcian blue in S. aureus V329, in contrast to what is seen in biofilm-negative mutant m556. Magnification, ×34,000.

FIG. 5.

Adherence of S. aureus strains to a mammary gland section. Magnification, ×200. A, lumen of mammary gland; B, epithelial layer; C, connective tissue.