Title : Identifying BBK32 sequences mediating the borrelia burgdorferi catch bond adhesion mechanism
Abstract:
Lyme disease is caused by spirochete members of the Borrelia burgdorferi (B. burgdorferi) sensu lato species complex and is transmitted by Ixodes ticks (Stanek et al., 2012). Hematogenous (blood-borne/vascular) dissemination is critical for the spread of B. burgdorferi from the initial tick bite site to other tissues including sites such as skin, joints, nervous system and heart, and for persistent infection (Wormser, 2006).
Adhesion of bacteria to the inner walls of blood vessels is a crucial step in this process, allowing the bacteria to slow down and escape the bloodstream into tissues where secondary infections can occur. Spirochete vascular interaction and extravasation is a multi-stage process initiated by short term tethering and dragging interactions (Moriarty et al., 2008). The initial step of interactions (tethering and dragging) is mediated by host plasma fibronectin (Fn) and glycosaminoglycans (GAGs), and by the Fn- and GAG- interacting B. burgdorferi adhesin BBK32 (Moriarty et al., 2012; Norman et al., 2008). An important barrier that inhibits but can also promote bacterial dissemination through the cardiovascular system is fluid shear stress. Mechanical forces derived from fluid shear stress greatly affect host-pathogen interactions. Catch bonds are interactions that are enhanced by the mechanical forces exerted on the ligand and its receptor (Sokurenko et al., 2008). Force-enhanced interactions such as catch bonds are key regulators of cellular interactions with vascular surfaces. It has shown that shear stress-regulated ligand-receptor interactions are also critical for bacterial dissemination in the cardiovascular system (Ebady et al., 2016).
To understand bacterial dissemination, it is critical to understand the physical mechanisms of bacterial-endothelial interactions under physiological shear stress not only at a molecular level, but in living cells. The development of a flow chamber system and tracking methods was crucial for studying the biomechanical and molecular mechanism of B. burgdorferi endothelial interactions under physiological shear stress conditions. In a recent study, the flow chamber model system using HUVECs was validated, and it showed that B. burgdorferi endothelial interactions recapitulate interaction conditions and properties observed in skin PCVs during IVM (Boczula et al., 2021; Ebady et al., 2016). The aim of this current study is to gain insight into bacterial-endothelial cell interactions in flow chambers under physiological shear stress through mapping the contributions of various bacterial mutants of B. burgdorferi. Using a panel of BBK32 internal deletion and truncation sequence variants spanning the full length of BBK32 and expressing these variants in recombinant form in E. coli and in non-adherent B. burgdorferi, will permit us to investigate the contributions of specific BBK32 sequences to the force-enhanced catch bond mechanism in our flow chamber model system and in in vitro studies performed with purified recombinant proteins and host ligands. Results show the possibility that sequences of the fibronectin binding region may be significant in dragging interactions.
