Staphylococcus aureus is now the leading cause of prosthetic heart valve infections. These infections are initiated with the formation of a physical bond between S. aureus and the surface of an implanted device. The colonizing bacteria form a biofilm that is resistant to antibiotics. Standard therapy is surgical removal of the infected device. This proposal will determine whether the binding force-signature of an S. aureus isolate can be used as a fundamental and practical indicator of pathogen-related risk for patients who are considering prosthetic device implants. We will focus on the bond between fibronectin (Fn), a host protein that coats prosthetic devices, and fibronectin-binding proteins (FnBPs) expressed on the cell wall of S. aureus. Our first Specific Aim will probe the practical meaning of the binding force-signature. We will develop a force-assay that could be used to identify potentially invasive S. aureus in the personal flora of patients who are candidates for a prosthetic cardiac device. Two hundred clinical isolates of S. aureus will be collected from four distinct populations of human subjects: patients with infected cardiac prostheses; patients with uninfected cardiac prostheses; patients undergoing hemodialysis; and a control group of healthy subjects lacking cardiac device implants. Clonal relationships of these isolates will be assessed with MLST and spa typing. Atomic force microscopy (AFM) will be used to probe the bond between a Fn-coated surface (i.e., substrate simulating a medical implant) and each of the 200 clinical isolates. Where possible, AFM will be used on isolates from distinct clonal lineages as determined by MLST and spa typing. The clinical isolates will be analyzed by PCR for the presence/absence of MSCRAMM genes, including fnbA which encodes FnBP. A Western blot will be used to verify the presence and estimate the amount of FnBP on the cell wall of each isolate. An optical microscope adhesion assay will count the number of cells for each isolate that attach to Fn-coated coverslips. Our second Specific Aim will probe the fundamental nature of the binding force-signature. We will examine the biomechanics of the in vivo bond between Fn and FnBP, and the underlying reason that some isolates form a more resilient bond. In addition to the AFM experiments discussed above, AFM will also be used to measure forces between (i) probes functionalized with the entire Fn protein or fragments of Fn (e.g., the 29 kD N-terminus) and (ii) isogenic strains/mutants that express truncated forms of FnBP, overexpress FnBP, or cannot express FnBP. AFM force spectra will be interpreted with the worm-like chain model to determine the length scale of unique sawtooth-like features observed in force curves. The length scale of the sawteeth will be compared to the primary sequence of FnBP, as determined by sequencing fnbA from 100 of the clinical isolates. Affinity maps will be generated on 25 strains by "tuning" into the force-signature of the Fn- FnBP bond thereby revealing the number and activity of FnBP on the surface of living cells of S. aureus. PUBLIC HEALTH RELEVANCE: A more complete understanding of the fundamental forces that permit Staphylococcus aureus to bind to prosthetic materials could provide important advances in the treatment and prevention of biofilm-associated infections of cardiac devices. Our preliminary work suggests that the "force taxonomy" of S. aureus may provide a fundamental yet practical indicator of pathogen-related risk for patients considering prosthetic device implants. [unreadable] [unreadable] [unreadable]