Structure of factor H-binding protein B (FhbB) of the periopathogen, Treponema denticola: insights into progression of periodontal disease.

Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A K(D) value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.

Crystallization of the factor H-binding protein, FhbB, from the periopathogen Treponema denticola.

Treponema denticola is a primary etiological agent of periodontal disease. T. denticola evades complement-mediated killing by binding to the host's factor H (FH), a negative regulator of the alternative complement pathway. The T. denticola FH-binding protein has been identified and designated as factor H-binding protein B (FhbB). Crystals of recombinant FhbB were obtained by the hanging-drop vapor-diffusion method using sodium citrate and 0.2 M sodium thiocyanate. FhbB crystals diffracted to 1.8 Šresolution and belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 46.76, c = 167.68 Å. Two FhbB molecules per asymmetric unit gave a Matthews coefficient of 2.2 Å(3) Da(-1) and a solvent content of 44%. FhbB is the smallest bacterially produced FH-binding protein identified to date. Determination of its structure will provide unique insight into the minimal structural determinants required for FH binding.

Analysis of a unique interaction between the complement regulatory protein factor H and the periodontal pathogen Treponema denticola.

Treponema denticola, a spirochete associated with periodontitis, is abundant at the leading edge of subgingival plaque, where it interacts with gingival epithelia. T. denticola produces a number of virulence factors, including dentilisin, a protease which is cytopathic to host cells, and FhbB, a unique T. denticola lipoprotein that binds complement regulatory proteins. Earlier analyses suggested that FhbB specifically bound to factor H (FH)-like protein 1 (FHL-1). However, by using dentilisin-deficient mutants of T. denticola, we found that T. denticola preferentially binds FH and not FHL-1, and that FH is then cleaved by dentilisin to yield an FH subfragment of approximately 50 kDa. FH bound to dentilisin-deficient mutants but was not cleaved and retained its ability to serve as a cofactor for factor I in the cleavage of C3b. To assess the molecular basis of the interaction of FhbB with FH, mutational analyses were conducted. Replacement of specific residues in widely separated domains of FhbB and disruption of a central alpha helix with coiled-coil formation probability attenuated or eliminated FH binding. The data presented here are the first to demonstrate the retention at the cell surface of a proteolytic cleavage product of FH. The precise role of this FH fragment in the host-pathogen interaction remains to be determined.

Comparative analysis of the properties and ligand binding characteristics of CspZ, a factor H binding protein, derived from Borrelia burgdorferi isolates of human origin.

Borrelia burgdorferi CspZ (BBH06/BbCRASP-2) binds the complement regulatory protein factor H (FH) and additional unidentified serum proteins. The goals of this study were to assess the ligand binding capability of CspZ orthologs derived from an extensive panel of human Lyme disease isolates and to further define the molecular basis of the interaction between FH and CspZ. While most B. burgdorferi CspZ orthologs analyzed bound FH, specific, naturally occurring polymorphisms, most of which clustered in a specific loop domain of CspZ, prevented FH binding in some orthologs. Sequence analyses also revealed the existence of CspZ phyletic groups that correlate with FH binding and with the relationships inferred from ribosomal spacer types (RSTs). CspZ type 1 (RST1) and type 3 (RST3) strains bind FH, while CspZ type 2 (RST2) strains do not. Antibody responses to CspZ were also assessed. Anti-CspZ antibodies were detected in mice by week 2 of infection, indicating that there was expression during early-stage infection. Analyses of sera collected from infected mice suggested that CspZ production continued over the course of long-term infection as the antibody titer increased over time. While antibody to CspZ was detected in several human Lyme disease serum samples, the response was not universal, and the titers were generally low. Vaccination studies with mice demonstrated that while CspZ is immunogenic, it does not elicit an antibody that is protective or that inhibits dissemination. The data presented here provide significant new insight into the interaction between CspZ and FH and suggest that there is a correlation between CspZ production and dissemination. However, in spite of its possible contributory role in pathogenesis, the immunological analyses indicated that CspZ is likely to have limited potential as a diagnostic marker and vaccine candidate for Lyme disease.

Complete Genome Sequence of Sphingobacterium sp. Strain ML3W, Isolated from Wings of Myotis lucifugus Infected with White Nose Syndrome.

Sphingobacterium sp. strain ML3W was isolated from the wing of a bat infected with white nose syndrome. We report the complete 5.33-Mb genome sequence of Sphingobacterium sp. strain ML3W, obtained using Pacific Biosciences technology. Being the second complete Sphingobacterium sequence, this will increase knowledge of the genus.

Tick salivary proteins offer the lyme disease spirochetes an easy ride and another way to hide.

The ability of the Lyme disease spirochetes to establish an infection in mammals is dependent in part on proteins of tick origin. Schuijt et al. (2011) investigate the role of the tick-derived protein, TSLPI, in spirochete transmission and in the evasion of killing by the lectin complement pathway.

Identification of the gene encoding the FhbB protein of Treponema denticola, a highly unique factor H-like protein 1 binding protein.

The gene encoding the Treponema denticola factor H-like protein 1 (FHL-1) binding protein, FhbB, was recovered and characterized. Sequence conservation, expression, and properties of FhbB were analyzed. The identification of FhbB represents an important step in understanding the contribution of FHL-1 binding in T. denticola pathogenesis and in development of periodontal disease.

Evidence that the BBA68 protein (BbCRASP-1) of the Lyme disease spirochetes does not contribute to factor H-mediated immune evasion in humans and other animals.

BBA68 (BbCRASP-1) of the Lyme disease spirochetes binds human factor H (FH) and FH-like protein 1 (FHL-1). Here we assess transcription of the BBA68 gene and production of BBA68 in infected mice and humans using real-time reverse transcriptase PCR and immunoblotting. The species specificity of FH binding to BBA68 was also tested. The data suggest that BBA68 does not play an important role in immune evasion in animals.

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.

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.

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.

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans.

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.

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.

Analysis of the OspE determinants involved in binding of factor H and OspE-targeting antibodies elicited during Borrelia burgdorferi infection in mice.

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.

Analysis of the ability of spirochete species associated with relapsing fever, avian borreliosis, and epizootic bovine abortion to bind factor H and cleave c3b.

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.

Evidence that the variable regions of the central domain of VlsE are antigenic during infection with lyme disease spirochetes.

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.

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.

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.

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 Borrelia burgdorferi 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.