John V. McDowell

Comprehensive Analysis of the Factor H Binding Capabilities of Borrelia Species Associated with Lyme Disease: Delineation of Two Distinct Classes of Factor H Binding Proteins

ABSTRACT Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing. Here we demonstrate significant differences in the fH binding capabilities of species of the Borreliaburgdorferi sensu lato complex. The percentages of B. burgdorferi, B. afzelii, and B. garinii bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively. The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes. Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the B. burgdorferi sensu lato complex.

Analysis of the OspE Determinants Involved in Binding of Factor H and OspE-Targeting Antibodies Elicited during Borrelia burgdorferi Infection in Mice

ABSTRACT Immune evasion by Lyme spirochetes is a multifactorial process involving numerous mechanisms. The OspE protein family undergoes antigenic variation during infection and binds factor H (fH) and possibly FHL-1/reconectin. In Borrelia burgdorferi B31MI, the OspE family consists of three paralogs: BBL39 (ErpA), BBP38, and BBN38 (ErpP). BBL39 and BBP38 are identical and therefore are referred to here as BBL39. The goals of this study were to assess the specificity of the antibody (Ab) response to the OspE paralogs and to identify the domains or determinants of OspE that are required for the binding of fH and OspE-targeting Abs that develop during infection. Here we demonstrate that at least some of the anti-OspE Abs produced during infection are paralog specific and that Ab binding requires conformational determinants whose formation requires both the N- and C-terminal domains of OspE. The binding of fH to OspE was also found to be dependent on conformational determinants. It is also demonstrated here that all of the OspE paralogs expressed by B. burgdorferi B31MI are capable of binding fH. The binding of fH to members of the OspF protein family was also assessed. In contrast to an earlier report, no binding of BBO39 or BBR42 to human fH was detected. Lastly, a series of competitive binding enzyme-linked immunosorbent assay analyses, designed to determine if fH and infection serum Abs bind to the same sites on OspE, revealed that these ligands interact with different regions of OspE.

Evidence That the Variable Regions of the Central Domain of VlsE Are Antigenic during Infection with Lyme Disease Spirochetes

ABSTRACT It has been postulated that the vls system of the Lyme disease spirochetes contributes to immune evasion through antigenic variation. While it is clear that vlsE undergoes sequence change within its variable regions at a high frequency during the early stages of infection, a definitive role in immune evasion has not been demonstrated. In this report we assessed the murine and human humoral immune response to recombinant (r)-VlsE variants that originally arose during infection in mice. Immunoblot analyses of r-VlsE variants were conducted by using serum samples collected from mice infected with Borrelia burgdorferi clones that carried different vlsE variants. All of the r-VlsE variants were recognized by infection sera regardless of the identity of the infecting clone or isolate. In addition, all variants were immunoreactive with a panel of human Lyme disease patient serum samples. It is evident from these analyses that the infection-induced VlsE variants share common epitopes that reside within conserved segments of these proteins. However, preabsorption experiments revealed that the variable regions of the central domain of VlsE, which undergo rapid mutation during infection, also influence the antigenic properties of the protein. A subset of the antibodies elicited against vlsE variants that differ in the sequences of their variable regions were found to be variant specific. Hence, in spite of a robust antibody response to conserved segments of VlsE, infection-induced sequence changes within the variable regions alter the antigenicity of VlsE. These results provide the first direct evidence of antigenic variation in the VlsE protein.

Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete Borrelia hermsii

In North America, tick-borne relapsing fever (TBRF) is caused by the spirochete species Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae. We previously demonstrated that some isolates of B. hermsii and B. parkeri are capable of binding factor H and that cell-bound factor H can participate in the factor I-mediated cleavage of C3b. Isolates that bound factor H expressed a factor H-binding protein (FHBP) that we estimated to be approximately 19 to 20 kDa in size and thus, pending further characterization, temporarily designated FHBP19. Until this report, none of the FHBPs of the TBRF spirochetes had been characterized. Here we have recovered the gene encoding the FHBP of B. hermsii YOR from a lambda ZAP II library and determined its sequence. The gene encodes a full-length protein of 22.7 kDa, which after processing is predicted to be 20.5 kDa. This protein, which we redesignate factor H-binding protein A (FhbA), is unique to B. hermsii. Two-dimensional pulsed-field gel electrophoresis and hybridization analyses revealed that the B. hermsii gene encoding FhbA is a single genetic locus that maps to a linear plasmid of approximately 220 kb. The general properties of FhbA were also assessed. The protein was found to be surface exposed and lipidated. Analysis of the antibody response to FhbA in infected mice revealed that it is antigenic during infection, indicating expression during infection. The identification and characterization of FhbA provides further insight into the molecular mechanisms of pathogenesis of the relapsing fever spirochetes.

Mutation and recombination in the upstream homology box-flanked ospE- related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection

ABSTRACT The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that theospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.

Analysis of the Ability of Spirochete Species Associated with Relapsing Fever, Avian Borreliosis, and Epizootic Bovine Abortion To Bind Factor H and Cleave C3b

ABSTRACT Some Borrelia species associated with Lyme disease bind the complement-regulatory protein factor H (fH), a process that may aid in immune evasion. In this report we demonstrate that some Borrelia species associated with relapsing fever bind fH, but not those associated with avian borreliosis and epizootic bovine abortion. Cell-bound fH was also found to mediate cleavage of exogenously supplied human C3b, demonstrating the biological relevance of fH binding and its possible importance in the pathogenesis of the relapsing-fever spirochetes.

Selective Binding of Borrelia burgdorferi OspE Paralogs to Factor H and Serum Proteins from Diverse Animals: Possible Expansion of the Role of OspE in Lyme Disease Pathogenesis

ABSTRACT The binding of Borrelia burgdorferi OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.

Analysis of Mechanisms Associated with Loss of Infectivity of Clonal Populations of Borrelia burgdorferi B31MI

ABSTRACT Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of Borrelia burgdorferi B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.

Demonstration of the Involvement of Outer Surface Protein E Coiled Coil Structural Domains and Higher Order Structural Elements in the Binding of Infection-Induced Antibody and the Complement-Regulatory Protein, Factor H

Factor H (fH) is an important regulator of the alternative complement cascade. Several human pathogens have been shown to bind fH to their surface, a process that facilitates immune evasion or cell to cell interaction. Among the pathogens that bind fH are some Borrelia species associated with Lyme disease and relapsing fever. The fH-binding proteins of the Lyme spirochetes form two classes (I and II). In Borrelia burgdorferi B31MI, class I includes the outer surface protein E (OspE) paralogs, L39, N38, and P38, whereas the class II group includes A68 and additional proteins that have not yet been identified. To identify the OspE determinants involved in fH and OspE-targeting infection-induced Ab (iAb) binding, deletion, random, and site-directed mutagenesis of L39 were performed. Mutations in several different regions of L39 abolished fH and or iAb binding, indicating that separable domains and residues of OspE are required for ligand binding. Some of the mutants that lost the ability to bind fH, iAb, or both had only a single amino acid change. Site-directed mutagenesis of three putative coiled coil motifs of OspE revealed that these higher order structures are required for fH binding but not for iAb binding. The data presented within demonstrate that the binding of fH and iAb to the OspE protein is mediated by higher order structures and protein conformation. These studies advance our understanding of fH binding as a virulence mechanism and facilitate ongoing efforts to use fH-binding proteins in the development of microbial vaccines.

Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site

Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.