Pathogens that traffic in the blood of their hosts must employ mechanisms to evade the host innate immune system, including the complement cascade. The Lyme disease spirochete,Borreliella burgdorferi, has evolved numerous outer membrane lipoproteins that interact directly with host proteins. Compared to Lyme disease-associated spirochetes, relatively little is known about how an emerging tick-borne spirochetal pathogen,Borrelia miyamotoi, utilizes surface lipoproteins to interact with a human host.B. burgdorferiexpresses the multifunctional lipoprotein, BBK32, that inhibits the classical pathway of complement through interaction with the initiating protease C1r, and also interacts with fibronectin using a separate intrinsically disordered domain.B. miyamotoiencodes two separatebbk32orthologs denotedfbpAandfbpB; however, the activities of these proteins are unknown. Here, we show thatB. miyamotoiFbpA binds human fibronectin in a manner similar toB. burgdorferiBBK32, whereas FbpB does not. FbpA and FbpB both bind human complement C1r and protect a serum-sensitiveB. burgdorferistrain from complement-mediated killing, but surprisingly, differ in their ability to recognize activated C1r versus zymogen states of C1r. To better understand the observed differences in C1r recognition and inhibition properties, high-resolution X-ray crystallography structures were solved of the C1r-binding regions ofB. miyamotoiFbpA and FbpB at 1.9Å and 2.1Å, respectively. Collectively, these data suggest that FbpA and FbpB have partially overlapping functions but are functionally and structurally distinct. The data presented herein enhances our overall understanding of how bloodborne pathogens interact with fibronectin and modulate the complement system.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borreliella burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway.BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of first component of complement, C1. To understand the structural basis for BBK32mediated C1r inhibition, we performed co-crystallography and size exclusion chromatographycoupled small angle x-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. The studies reported here, for the first time, provide a structural basis for classical pathway-specific inhibition by a human pathogen.