AVS 51st International Symposium
    Biomaterial Interfaces Monday Sessions
       Session BI-MoP

Paper BI-MoP22
Investigation of the Binding Mechanism of the Bacterial Adhesin Scp to Fibronectin

Monday, November 15, 2004, 5:00 pm, Room Exhibit Hall B

Session: Poster Session
Presenter: J.R. Hull, The University of Washington
Authors: J.R. Hull, The University of Washington
D.G. Castner, University of Washington
G. Tamura, Children's Hospital and Regional Medical Center
Correspondent: Click to Email
Several pathogens bind specifically to immobilized fibronectin (Fn), and not to soluble Fn, including Group B Streptococci (GBS), S. pneumoniae, S. sanguis, and p-fimbriated strains of E. coli. However, the structural basis for the specific adherence of Scp to immobilized Fn has not been resolved. There are two possible mechanisms for Scp binding to Fn. First, specific conformational determinants of Fn that allow Scp binding may be unmasked when Fn is immobilized. Second, Scp may bind to a combinatorial determinant formed by the clustering of multiple immobilized Fn molecules. The Scp-Fn binding interaction is being studied with two complementary analysis methods. Surface Plasmon Resonance (SPR) is being used to study the kinetics of the binding interactions and Atomic Force Microscopy (AFM) is being used to investigate single molecule interactions. Supporting experiments to determine surface compositions was done using X-ray Photoelectron Spectroscopy. SPR has shown that the binding affinity of soluble Scp to adsorbed Fn is approximately nano-molar and that Scp does not bind soluble Fn. A model system was set up to develop AFM methods for examining the Scp-Fn interactions. Collagen related peptides (CRPs) on the AFM tip were used to interact with adsorbed Fn. The CRPs were attached to the AFM tip via homo-functional N-hydroxysuccinimide (NHS) poly(ethylene glycol) (PEG) cross-linker. The jump heights of the force distance curves were graded based on Studen@aa t@s t-test and only events with a nominal grade were further analyzed. Analysis of unbinding events shows that there are multiple interactions centered at 75 pN and there are multiple length scales over which these interactions occur. There are at three length scales over which these interactions occur: 5 nm which is attributable to nonspecific adhesion, 15 nm which is the NHS-PEG spacer length, and 30 nm and greater which is attributable to CRP-Fn interactions.