The dual activity of BBK32: Implications for simultaneous inhibition of borrelial-specific antibody-dependent complement activation and fibronectin binding

Abstract

Complement inhibition is exploited by extracellular pathogens to combat clearance. Borreliella burgdorferi, the causative agent of Lyme disease, harnesses complement evasion techniques to establish and maintain infection in mammalian hosts. B. burgdorferi encodes bbk32, a surface lipoprotein that binds extracellular matrix (ECM) components, specifically glycosoaminoglycans (GAGs) and fibronectin (Fn) within its amino terminus. In addition to its ECM-binding functions, the carboxy terminal half of BBK32 binds to the C1r protease and prevents complement activation. This classical complement inhibitory activity protects B. burgdorferi from complement-mediated killing following the addition of normal human serum. Herein we demonstrate that full length BBK32 binds both Fn and C1 concurrently, indicating that binding of these macromolecules do not sterically hinder their simultaneous interaction. Given the link of antibody dependence to the classical pathway, we tested how the presence of BBK32 would protect B. burgdorferi from antibody mediated, complement dependent killing. The presence of BBK32 provided protection against borrelial-specific antibody binding and concomitant complement activation in vitro. We also demonstrated, using both fluorescence microscopy and flow cytometry, that the presence of BBK32 was required for reduced C4 deposition on the surface of borrelial cells. This work demonstrates the potential for BBK32 to simultaneously bind to both C1r and Fn and contributes to the broader understanding of the ability of B. burgdorferi to evade antibody-dependent complement-mediated killing. We contend that these observations ostensibly provide B. burgdorferi with coincident dissemination and immune evasion activities needed for optimal survival during infection. AUTHOR SUMMARYLyme disease, caused by Borreliella burgdorferi and other related species, is the most common arthropod-borne infection in the United States. As an extracellular pathogen, B. burgdorferi is exposed to the complement system--a soluble proteolytic cascade that clears invaders. Complement is defined by three pathways known as the alternative, lectin, and classical. The classical complement cascade is activated by the binding of antibodies to a foreign or damaged cell. For B. burgdorferi, the BBK32 surface protein is known to mute the classical pathway by binding and inhibiting the initiating protease C1r. However, no studies have addressed how BBK32 protects infectious B. burgdorferi from borrelial-specific antibody binding and clearance. Here we show that native BBK32 protects infectious B. burgdorferi from antibody-dependent, complement mediated killing. Given BBK32s other activity--namely its known adherence to fibronectin--we were interested to test if BBK32 could bind the C1 complex, which contains C1r, together with fibronectin. Our results suggest that these complex macromolecules can bind BBK32 simultaneously. These observations suggest that the dual activity of BBK32, namely fibronectin binding and C1r inhibition, are not mutually exclusive and contribute to B. burgdorferis ability to establish infection and evade antibody-based host clearance, respectively.