Danielle Pischulla

Background: Lyme Disease is a multisystem illness with the potential to affect the skin, joints, heart, and nervous system and accounts for roughly 90% of all vector-borne diseases in the United States.^1,2 The spirochete bacteria, Borrelia burgdorferi, causes Lyme Disease and is transmitted by the bite of the Ixodes tick. Currently, there is no preventative vaccine or reliable diagnostic test for Lyme Disease due partially to the complexity of the Borrelia genome and presence of antigenic variation.³ Previously, BBK32 was thought only to play a role as a fibronectin and glycosaminoglycan-binding protein aiding in vascular endothelial attachment.^3,4 Characterization of BBK32 at its C terminus revealed its role as a multifunctional protein that aids in the first phase of tissue dissemination, bloodstream survival. Further exploration of BBK32 as a multifunctional protein could elucidate capabilities for diagnosis and treatment of Lyme Disease as well as new therapeutic options to reduce the overactivation of the classical complement cascade in several pathologies and the collateral damage that can occur as a result.⁵

Research Objectives: This narrative review explores the mechanisms BBK32-C uses to inhibit C1/C1r activity in the classical complement pathway.

Methods: An online search in the database PubMed was conducted from 2016 to 2023 using the following keywords: “Lyme Disease,” “BBK32,” “BBK32-C,” “pathogenesis,” and “therapy.”

Results: A study found a novel mechanism that indicated BBK32-C binds C1 specifically and tightly in a calcium-dependent manner preventing the initiation of the classical complement pathway.⁴ Further investigation showed BBK32-C also recognizes and binds C1r, preventing autoactivation of C1r and subsequent cleavage of C1s proenzyme through an assumed mechanism of noncovalent inhibition of the C1r enzyme, trapping C1 in its zymogen form.⁴ Characterization of BBK32-C’s structure revealed the C terminus is positively charged and composed of an antiparallel, four alpha-helix bundle.⁶ Analysis of the C1/C1r binding site determined BBK32 only recognizes and binds C1r fragments containing a serine protease (SP) domain; without an SP domain, C1r cannot have a high affinity interaction with BBK32-C.⁶ Analysis of Borrelia genospecies revealed B. burgdorferi and B. afzelii have C1/C1r inhibition ability whereas B. garinii does not. This suggests some of the non-conserved amino acid substitutions between orthologs are necessary for C1/C1r inhibitory activity.⁶ Development of a model of BBK32-C mediated inhibition of C1/C1r determined BBK32-C’s binding site is very close in proximity to C1r’s catalytic site, residues R248 and K327 play a major role in the ability for BBK32 to inhibit C1/C1r, and BBK32 interacts with non-conserved loop regions while simultaneously occluding C1r subsites, S1 and S1’.⁷ In a clinical application of BBK32, a diagnostic test was developed using a chimeric BBK32 protein that was highly reactive to IgG antibodies in the serum of infected individuals with no cross-reactivity with healthy serum.⁸

Conclusion: Studies have found BBK32-C has multiple mechanisms by which it targets and inhibits C1/C1r. BBK32 has an unrecognized therapeutic potential that if studied further could be a candidate for a preventative vaccine or utilized as a therapeutic mechanism to combat autoimmune disorders involving the overaction of the classical complement system.

Works Cited:

1. Kullberg BJ, Vrijmoeth HD, van de Schoor F, Hovius JW. Lyme borreliosis: diagnosis and management. BMJ. 2020;369:m1041. Published 2020 May 26. doi:10.1136/bmj.m1041
2. Radolf JD, Caimano MJ, Stevenson B, Hu LT. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol. 2012;10(2):87-99. Published 2012 Jan 9. doi:10.1038/nrmicro2714
3. Coburn J, Garcia B, Hu LT, Jewett MW, Kraiczy P, Norris SJ, Skare J. Lyme Disease Pathogenesis. Current Issues in Molecular Biology. 2021; 42(1):473-518. doi.org/10.21775/cimb.042.473
4. Garcia BL, Zhi H, Wager B, Höök M, Skare JT. Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex. PLoS Pathog. 2016;12(1):e1005404. Published 2016 Jan 25. doi:10.1371/journal.ppat.1005404
5. Barnes AP, Khandelwal S, Sartoretto S, et al. Minimal role for the alternative pathway in complement activation by HIT immune complexes. J Thromb Haemost. 2022;20:2656-2665. doi: 10.1111/jth.15856
6. Xie J, Zhi H, Garrigues RJ, Keightley A, Garcia BL, Skare JT. Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes. PLoS Pathog. 2019;15(3):e1007659. Published 2019 Mar 21. doi:10.1371/journal.ppat.1007659
7. Garrigues RJ, Powell-Pierce AD, Hammel M, Skare JT, Garcia BL. A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes. J Immunol. 2021;207(11):2856-2867. doi:10.4049/jimmunol.2100815
8. Grąźlewska W, Ferra B, Rudzińska M, Holec-Gąsior L. Borrelia burgdorferi BmpA-BBK32 and BmpA-BBA64: New Recombinant Chimeric Proteins with Potential Diagnostic Value. Pathogens. 2021;10(6):767. Published 2021 Jun 18. doi:10.3390/pathogens10060767