Functional characterization of BbCRASP‐2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL‐1

Abstract

SummaryBorrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complementmediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal–tick infection cycle

“…The B. afzelii BaCRASP-1 is also encoded by an orthologous gene, likewise located on its lp54 homolog . The cspZ gene, encoding BbCRASP-2, is located on another linear DNA element, lp28-3, but is not closely related to any other gene within the B. burgdorferi genome (Fraser et al, 1997;Casjens et al, 2000;Hartmann et al, 2006). Orthologous genes have been identified in all other Lyme disease spirochete genospecies, although it is not yet clear whether or not the encoded proteins bind factor H and FHL-1 (Rogers and Marconi, 2007, and our unpublished results).…”

“…B. burgdorferi produces up to five different BbCRASPs, which are reviewed herein. At least two of the BbCRASPs contribute to complement resistance in vitro (Brooks et al, 2005;Hartmann et al, 2006) but the question of why B. burgdorferi produces multiple distinct factor H-binding proteins remains unsolved. Confounding matters further, both wild-type and factor H-deficient mice can be infected by Lyme disease spirochetes to equal degrees , suggesting that factor H-binding is not essential for efficient mammalian infection.…”

“…produces several different outer surface proteins collectively termed CRASPs (complement regulator-acquiring surface proteins). These lipoproteins share affinities for the host fluid phase negative regulators of complement factor H and/or FHL-1 (factor H-like protein 1) (Hellwage et al, 2001;Kraiczy et al, 2001Kraiczy et al, , 2003Kraiczy et al, , 2004aKraiczy et al, , 2004bAlitalo et al, 2002Alitalo et al, , 2005Stevenson et al, 2002;McDowell et al, 2003;Hartmann et al, 2006;Kraiczy and Würzner, 2006;Herzberger et al, 2007). Those two host proteins promote breakdown of C3b and inactivation of the alternative pathway C3 convertase (Janeway et al, 1999;Kraiczy and Würzner, 2006).…”

Dual Binding Specificity of a Borrelia hermsii-Associated Complement Regulator-Acquiring Surface Protein for Factor H and Plasminogen Discloses a Putative Virulence Factor of Relapsing Fever Spirochetes

“…hermsii (ATCC35209) strain HS1 and YOR isolates (provided by T. Schwan, Rocky Mountain Laboratories) and the Lyme disease spirochetes B. burgdorferi isolate B31 and mutant B313 were cultivated in BarbourStoenner-Kelly (BSK)-H complete medium (PAN Biotech) supplemented with 5% rabbit serum (Cell Concept) at 30°C. B313 mutant spirochetes harbor plasmids cp32-1, cp32-2, cp32-3, cp32-4, cp26, and lp7 exclusively and therefore lack expression of BbCRASP-1 to BbCRASP-4 (27). Bacteria were harvested by centrifugation and washed with PBS.…”

Biology of Infection with Borrelia burgdorferi

“…Considerable attention has been paid to several B. burgdorferi proteins that co-opt the complement regulators factor H and factor H-like proteins, which normally protect mammalian cells from attack by their own complement by blocking activation of the alternative pathway [136][137][138][139][140][141]. Surface coating with factor H could protect the bacteria from killing or opsonization by host complement, facilitating infection.…”