Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

Abstract

SummaryThe ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and dif®culties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature-and F-actindependent manner. Formaldehyde-®xed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in noninvasive strains conferred invasiveness; and (iii) the soluble isolated ®bronectin-binding domain of FnBP (D1±D4) completely blocked invasion. Integrin a 5 b 1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble ®bronectin. FnBP-de®cient mutants lost invasiveness for epithelial cells, endothelial cells and ®broblasts. Thus, ®bronectin-dependent bridging between S. aureus FnBPs and host cell integrin a 5 b 1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.

Analysis of prophages harbored by the human-adapted subpopulation of Staphylococcus aureus CC398

“…Strain KH11 was used as a non-invasive, negative control. The internalization procedure and the lysostaphin protection assay were based on a published method (Sinha et al, 1999) with minor differences. Briefly, cultures of the cell line 293 were maintained under humidified air with 5% CO 2 at 37°C in Dulbecco's minimal essential medium (DMEM)-nutrient mixture F-12 (nut mix F-12) containing Glutamax I, a stable glutamine dipeptide, supplemented with 10% fetal calf serum (FCS), 50 IU of penicillin per mL, and 50 lg of streptomycin per mL.…”

“…For the lysostaphin assay, 293 cells were plated in poly-ornithine coated plates (1.8 cm diameter) and cultured for 24 h, such that there were approximately 0.9 Â 10 5 cells/well. Bacterial were harvested from culture, washed, counted and diluted (Sinha et al, 1999); these bacterial dilutions were added to the plates containing 293 cells at a multiplicity of infection [MOI] of 15:1. The plates were incubated for 1 h at 37°C, and the bacterial suspensions were then replaced with lysostaphin medium (DMEM-nut mix F-12, 10% FCS, 20 lg of lysostaphin per mL).…”

“…aureus is not an obligatory intracellular pathogen. However, the intrinsic virulence of isolates is associated with their ability to reside in the cytoplasm of host epithelial or endothelial cells, particularly in chronic infections (Sinha et al, 1999;Voyich et al, 2005). Invasion of epithelial cells by representative isolates (the parental (S123 and S124) and donor (S100) strains, LA isolates (S1 and S130), non-LA isolates (S89 and S94), and two control strains (Cowan and KH11) was evaluated.…”

Cell adhesion receptors - signaling capacity and exploitation by bacterial pathogens

“…A similar strategy is seen in Staphylococcus aureus, where surface proteins (fibronectin binding proteins, FnBPs) mediate the binding of the ECM protein fibronectin, which in turn leads to recognition by the cellular fibronectin receptor, integrin a5b1 [38,55]. As invasion can be blocked by specific antibodies against integrin a5b1 or cytochalasin D, S. aureus seems to utilize the integrin-actin cytoskeleton linkage to gain access to the interior of the cell.…”

Staphylococcus aureus colonization and non-influenza respiratory viruses: Interactions and synergism mechanisms

“…The fibronectin-binding protein A or B (FnBPA/B) are S. aureus cell wall-anchored Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) that bind type I motif of fibronectin through a tandem beta-zipper interaction [58]. The α5β1 integrin expressed on host cellmembrane binds the RGD motif of fibronectin [59]. Fibronectin is thus acting as a bridge between S. aureus and the host cell, and the complex formed by FnBPA/B, fibronectin and α5β1 integrin leads to S. aureus internalization inside the epithelial cell [57].…”