Quantification of staphylococcal adhesion using optical tweezers
Simpson, Kathryn Hicks
Doctor of Philosophy
Biofilm formation, a common cause of medical device failure and tissue infection, often follows bacterial adhesion to proteins present in the tissue or adsorbed on the implant surface. Certain species of gram-positive bacteria have covalently anchored transmembrane molecules called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which mediate adhesion. Developing a greater understanding of the interactions between MSCRAMMs and their ligands can lead to improved methods of combating bacterial adhesion. The goal of this research was to use optical tweezers to quantify and characterize the forces of staphylococcal detachment from surfaces coated with extracellular matrix (ECM) molecules. An optically trapped bacterium was brought in contact with an ECM-coated polystyrene microsphere, and the force required to separate the cell and microsphere was determined. The forces required to detach S. epidemidis from fibronectin occurred in a series of clusters whose means were integer multiples of an 18-piconewton (pN) base value depending on the number of bonds formed. For S. aureus binding, this estimated single-bond force was 25 pN for fibronectin and 20 pN for fibrinogen, respectively. In S. aureus, we have found that varying degrees of mutation of the fibronectin-binding MSCRAMM may cause reduction or inhibition of binding depending on the degree of mutation. However, multiple mutations are required before any reduction in binding is observed, which confirms that multiple regions of the S. aureus fibronectin MSCRAMM may substitute for one another in the binding process. We have also tracked the force required to detach S. aureus from fibronectin using the signal generated by the trapped cell on a quadrant photodiode throughout the detachment process for a series of loading rates. The magnitude of the force increased in an approximately linear fashion until the point of bond rupture. The peak single-bond rupture forces ranged from 10 to 29 pN for loading rates spanning three orders of magnitude from 101 to 103 pN/s. Bond lifetime increased with loading rate, which suggests the presence of a catch-bonding mechanism. This work provides additional insight into the specific binding mechanisms of staphylococci and is a step toward developing improved methods of preventing or treating infections.
Microbiology; Biomedical engineering; Biophysics