Fibronectin binding protein A-mediated cellular infections by Staphylococcus aureus (Rosenbach, 1884)

Abstract

Staphyloccous aureus is one of the leading pathogens in nosocomial infections and can cause life threatening diseases such as bacteremia and acute endocarditis. The surface adhesin fibronectin binding protein A (FnBPA) mediates attachment to the host cell extracellular matrix component fibronectin (Fn) and subsequent actin-dependent invasion. Bacteria-associated Fn is mainly bound to host cell integrins. While the region of Fn responsible for binding FnBPA is well defined, the domains that bind the host cell which lead to FnBPA-mediated adherence and uptake are unclear. In addition, a role for the maturation state of Fn in FnBPA-mediated infection has not been explored. To answer these questions, we generated Fns harboring inactivating mutations in the central cell-binding RGD motif and adjacent synergy site, or purified proteolytic Fn fragments, comprising different parts of the N-terminal region. In their soluble form, bound to FnBPA-particles, these mutants or fragments were reacted with Fn-deficient cells or, in their cellularly organized form, were reacted with uncoated FnBPA-particles. For soluble Fn, the RGD motif was important for adhesion to and invasion of cells by FnBPA-particles. The synergy site was, however, important for adhesion but not invasion. Additionally, Fn 70 kDa but not Fn 30 kDa promoted invasion, suggesting a key role for the gelatin-binding domain in Fn RGD-independent invasion. In sharp contrast, the organization of Fn into fibrils reduced overall invasion and, under these conditions, neither the RGD motif nor the synergy site were important for adhesion to and invasion of cells by FnBPA-particles. Thus, we demonstrate important roles for the central cell-binding domain and the maturation state of Fn in the infection mechanism of S. aureus. To further analyze the connection of mutant Fns to the integrin-linked cytoskeleton, the cell surface motility and adhesive structures formed by bacteria coated with different Fns were compared. Bacteria coated with either RGD-mutated Fn, synergy site-mutated Fn or Fn 70 kDa showed a reduced displacement and less direct transport on the cell surface. In addition, when the RGD motif was mutated, proteins important for integrin-dependent signaling and the linkage of integrins to the cytoskeleton were not recruited. These experiments show that defects in the central cell-binding domain of Fn impair the connection of integrins to the actin cytoskeleton. The results presented here further our understanding of the mechanisms by which S. aureus infect the host.