Abstract
The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex.
Highlights
Spirochetes belonging to the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis and include B. burgdorferi sensu stricto, B. garinii, and B. afzelii
We demonstrated that the B. burgdorferi protein BBK32 blocks the activation of the classical pathway of complement by binding to and inhibiting the initiating protease, C1r
We show that the BBK32 orthologue from B. garinii, designated BGD19, is unable to neutralize complement-mediated serum killing
Summary
Spirochetes belonging to the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis and include B. burgdorferi sensu stricto, B. garinii, and B. afzelii. B. burgdorferi sensu stricto (referred to as B. burgdorferi hereafter) causes 300,000 cases of Lyme disease in the United States each year [1], while B. garinii and B. afzelii are the most common etiological agent of Lyme disease in Europe and Asia [2]. Complement activation is initiated by soluble pattern recognition proteins which are capable of discerning foreign molecular surfaces. The classical pathway is activated upon binding of the complement protein C1q to antigen-bound antibodies (i.e. immune complexes). Independent of the molecular initiating event, all three pathways proceed by activation of a series of specialized serine proteases that converge on the central molecule of complement, C3. C3 is cleaved by enzymatic complexes called C3 convertases which results in complement amplification at the target
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