[Evaluation of significance of Borrelia garinii spirochete recombinant protein DbpB for serodiagnostics of ixodes tick-borne borreliosis].

AIM: Obtaining recombinant protein DbpB of West Siberia Borrelia garinii 20047 isolate and evaluation of its antigen activity for the possible use in serodiagnostics of ixodes tick-borne borreliosis (ITB). MATERIALS AND METHODS: Coding region of dbpB gene of novosibirsk B. garinii 20047 isolate was amplified by PCR and cloned as part of expressing pETm vector in Escherichia coli BL21 (DE3) strain cells. Recombinant protein DbpB produced by the selected clone was studied by EIA method for its ability to react with sera antibodies of ITB patients. RESULTS: E. coli BL21 (DE3) clone producing recombinant protein DbpB in quantity of 30% of total E. coli cell protein was obtained. Homology of amino acid sequence of recombinant protein DbpB of novosibirsk B. garinii 20047 isolate with primary structures of B. garinii, B. afzelii and B. burgdorferi sensu stricto spirochete genospieces DbpB proteins presented in GenBank database was 98.4, 77 and 73%, respectively. Sensitivity of immune enzyme detection in sera of ITB patients with migrating erythema of IgM and IgG reacting with DbpB antigen was 13.9 and 20.0%, respectively. Frequency of detection of IgM and IgG against DbpB in patient sera with disseminated ITB form was 15.7 and 43.8%, respectively. Specificity of immune enzyme detection of antibodies against recombinant antigen DbpB in which sera of syphilis, rheumatoid arthritis patients and healthy donors used as control sera was 100%. CONCLUSION: DbpB recombinant protein of novosibirsk B. garinii 20047 isolate may be used as one of antigens for highly specific serodiagnostics of ITB disseminated stage.

Identification and functional characterisation of Complement Regulator Acquiring Surface Protein-1 of serum resistant Borrelia garinii OspA serotype 4.

BACKGROUND: B. burgdorferi sensu lato (sl) is the etiological agent of Lyme borreliosis in humans. Spirochetes have adapted themselves to the human immune system in many distinct ways. One important immune escape mechanism for evading complement activation is the binding of complement regulators Factor H (CFH) or Factor H-like protein1 (FHL-1) to Complement Regulator-Acquiring Surface Proteins (CRASPs). RESULTS: We demonstrate that B. garinii OspA serotype 4 (ST4) PBi resist complement-mediated killing by binding of FHL-1. To identify the primary ligands of FHL-1 four CspA orthologs from B. garinii ST4 PBi were cloned and tested for binding to human CFH and FHL-1. Orthologs BGA66 and BGA71 were found to be able to bind both complement regulators but with different intensities. In addition, all CspA orthologs were tested for binding to mammalian and avian CFH. Distinct orthologs were able to bind to CFH of different animal origins. CONCLUSIONS: B. garinii ST4 PBi is able to evade complement killing and it can bind FHL-1 to membrane expressed proteins. Recombinant proteins BGA66 can bind FHL-1 and human CFH, while BGA71 can bind only FHL-1. All recombinant CspA orthologs from B. garinii ST4 PBi can bind CFH from different animal origins. This partly explains the wide variety of animals that can be infected by B. garinii.

Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.

Lyme borreliosis, the most common vector-borne illness in Europe and the United States, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex and transmitted by Ixodes ticks. In humans, the spirochetes disseminate from the tick bite site to multiple tissues, leading to serious clinical manifestations. The ability of spirochetes to survive in ticks during blood feeding is thought to be essential for Lyme borreliae to be transmitted to different vertebrate hosts. This ability is partly attributed to several B. burgdorferi proteins, including BBA52 and Lp6.6, which promote spirochete survival in nymphal ticks feeding on mice. One of the strategies to identify such proteins without using live animals is to feed B. burgdorferi-infected ticks on blood via artificial feeding chambers. In previous studies, ticks were only fed on bovine blood in the feeding chambers. In this study, we used this chamber model and showed that I. scapularis ticks will not only acquire bovine blood but human and quail blood as well. The latter two are the incidental host and an avian host of Lyme borreliae, respectively. We also investigated the roles that BBA52 and Lp6.6 play in promoting spirochete survival in nymphal ticks fed on human or quail blood. After feeding on human blood, spirochete burdens in ticks infected with an lp6.6-deficient B. burgdorferi were significantly reduced, while bba52-deficient spirochete burdens in ticks remained unchanged, similar to the wild-type strain. No strain showed a change in spirochete burdens in ticks fed on quail blood. These results indicate that Lp6.6 plays a role for B. burgdorferi in nymphs fed on human but not quail blood. Such information also demonstrates that the artificial feeding chamber is a powerful tool to identify B. burgdorferi proteins that promote vertebrate host blood-specific spirochete survival in I. scapularis ticks.

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering.

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.

Evidence for an alpha-helical epitope on outer surface protein A from the Lyme disease spirochete, Borrelia burgdorferi: an application of steady-state and time-resolved fluorescence quenching techniques.

Outer surface protein A (OspA) is a major antigen of Borrelia burgdorferi, the etiological agent of Lyme disease. A recombinant form of OspA (OspA-257) from B. burgdorferi, strain B31, contains 257 amino acids and a single tryptophan residue at position 216 (Trp-216). Mapping studies indicate that Trp-216 is involved in the epitope for the agglutinating monoclonal antibody 105.5. However, the fluorescence emission maximum of the native protein is 330 nm, indicating that Trp-216 is not solvent-exposed. Primary structure analysis suggests an alpha-helical conformation for residues approx. 204-217, which, if located on the protein surface, would allow Trp-216 to be buried, while leaving hydrophilic residues on the opposite side of the helix exposed. This helix would place Lys-212 within approx. 6 A of Trp-216; the presence of such a positively-charged residue can, in principle, be ascertained from fluorescence quenching studies. Stern-Volmer plots confirm that Trp-216 is indeed buried in the native protein, but is readily accessible to the small polar quencher, acrylamide. Furthermore, the dominant component of the fluorescence emission shows only weak dynamic quenching by the positively-charged quencher, Cs+, while the minor component undergoes static quenching by I-, indicating the proximity of a positively-charged residue. These data are consistent with the existence of an alpha-helix from residues 204-217 in the predicted orientation at the protein surface, hence indicating the structure of the antigenic determinant.

Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy.

The etiological agent of Lyme disease is the tick-borne spirochete, Borrelia burgdorferi. A major antigen of B. burgdorferi is a 31 kDa lipoprotein called outer surface protein A (OspA). Recently, a truncated form of OspA (lacking 17 amino acids at the N-terminus) was cloned, expressed and purified in large quantities (Dunn, J.J., Lade, B.A. and Barbour, A.G. (1990) Protein Expression and Purification 1, 159-168). The truncated protein (OspA-257) is water-soluble, retains the ability to bind antibodies from the sera of Lyme disease patients and may prove useful in development of a vaccine against Lyme disease. We have used far UV circular dichroism (CD) and fluorescence spectroscopy to characterize the secondary structure of and to study conformational changes in OspA-257. CD spectra from 260 to 178 nm predict five classes of secondary structure: alpha-helix (11%), anti-parallel beta-sheet (32%), parallel beta-sheet (10%), beta-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for alpha-helical regions (residues 100-107, 121-134, 253-273) and for antigenic determinants (Lys-46, Asp-82, Lys-231). CD spectra of the native protein show little change from pH 3 to 11. Thermal denaturation curves, indicate that 'salt bridges' play a role in stabilizing the native protein. Both thermal and chemical denaturations that eliminate all secondary structure as judged by CD or fluorescence are reversible. Denaturation by guanidine-HCl (gdn-HCl) appears to be a cooperative, two-state transition, as indicated by a sudden change in the CD spectrum at approximately 0.75 M gdn-HCl, and an isodichroic point at 208 nm in all CD spectra measured from 0.0-1.75 M gdn-HCl.

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet.

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain.

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.

Structural identification of a key protective B-cell epitope in Lyme disease antigen OspA.

Outer surface protein A (OspA) is a major lipoprotein of the Borrelia burgdorferi spirochete, the causative agent of Lyme disease. Vaccination with OspA generates an immune response that can prevent bacterial transmission to a mammalian host during the attachment of an infected tick. However, the protective capacity of immune sera cannot be predicted by measuring total anti-OspA antibody. The murine monoclonal antibody LA-2 defines an important protective B-cell epitope of OspA against which protective sera have strong levels of reactivity. We have now mapped the LA-2 epitope of OspA using both NMR chemical-shift perturbation measurements in solution and X-ray crystal structure determination. LA-2 recognizes the three surface-exposed loops of the C-terminal domain of OspA that are on the tip of the elongated molecule most distant from the lipid-modified N terminus. The structure suggests that the natural variation at OspA sequence position 208 in the first loop is a major limiting factor for antibody cross-reactivity between different Lyme disease-causing Borrelia strains. The unusual Fab-dominated lattice of the crystal also permits a rare view of antigen flexibility within an antigen:antibody complex. These results provide a rationale for improvements in OspA-based vaccines and suggest possible designs for more direct tests of antibody protective levels in vaccinated individuals.

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human factor-H and is predicted to form a membrane-spanning ß-barrel.

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane, is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The outer membrane protein A (OmpA) domain is an eight-stranded membrane-spanning ß-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work, we describe the identification of four novel OmpA-like ß-barrels from Borrelia afzelii, the most common cause of erythema migrans (EM) rash in Europe. Structural characterization of one these proteins (BAPKO_0422) by SAXS and CD indicate a compact globular structure rich in ß-strand consistent with a monomeric ß-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25×45 Å (1 Å=0.1 nm). Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal phenylalanine residue commonly found in Proteobacterial OM proteins is replaced by isoleucine/leucine or asparagine. BAPKO_0422 is demonstrated to bind human factor H (fH) and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31.

Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli sigma 70 and Bacillus subtilis sigma 43 showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.

Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking.

Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed 'semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis.

FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria.

Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

Lyme disease in Taiwan: primary isolation of Borrelia burgdorferi-like spirochetes from rodents in the Taiwan area.

To investigate the prevalence of Lyme disease infection in Taiwan, we conducted a zoonotic survey for spirochetal infection in the small mammals. Ear tissues of trapped rodents collected from various localities in Taiwan were incubated into BSK-H culture medium and examined for the evidence of spirochetal infection by dark-field microscopy. Spirochetes cultured from six species of wild and peridomestic rodents and seven isolates, designated TWKM 1-7, were purified by serial dilution and membrane filtration. Infection was detected in 16.6% (53 of 320) of captured rodents and the highest infection rate (36.4%) was observed in the brown country rat (Rattus losea, Swinhoe). Higher infection rates based on the geographic distribution were observed in the eastern localities and on Kimmen Island. Reactivity with Borrelia burgdorferi-specific monoclonal antibodies and Western blot analysis indicated that these Taiwan isolates were closely related to the causative agent of Lyme disease, B. burgdorferi sensu lato. These results provide the first evidence of the existence of Lyme disease spirochetes in the Taiwan area.

Comparison of Borrelia isolated from UK foci of Lyme disease.

Restriction endonuclease digestion of linear borrelial chromosomal DNA showed that three isolates of UK Lyme disease spirochaetes differed markedly from each other and from published data for other isolates from North America and continental Europe. Analysis of linear plasmid bands revealed that UK isolates differed from each other in the number and sizes of the plasmids in isolates from different foci of UK Lyme disease. Fatty acid analysis (of fatty acid methyl ester (FAME) profiles) showed the UK isolates clustering together with the relapsing fever spirochaetes, Borrelia turicatae and Borrelia parkeri. These data are discussed in respect of current knowledge of Lyme borreliosis in the UK.

Molecular characterization of the p100 gene of Borrelia burgdorferi strain PKo.

The p100 gene coding for the p100 protein of Borrelia burgdorferi strain PKo has been cloned, sequenced and expressed in Escherichia coli. An open reading frame including upstream and downstream sequences with potential translation and transcription signals could be identified. The reading frame consists of 1989 nucleotides corresponding to a protein of 663 amino acids and a calculated molecular mass of 75.8 kDa. The protein has a leader peptide and is processed without modification at the N-terminus. A high percentage of amino acid sequence identity could be found to the high-molecular mass protein p83/p93 of B. burgdorferi strain B31.

Evidence that Borrelia burgdorferi immunodominant proteins p100, p94 and p83 are identical.

Recently there have been reports on high-molecular mass components of Borrelia burgdorferi, namely the p100, p94 and p83, which claimed these proteins to be specific marker antigens for the serodiagnosis of late Lyme borreliosis. The nucleotide sequences of the p100 and p83 have been published. The alignment of the deduced N-terminal amino acid sequences with the N-terminal sequence of the p94 now provides evidence that all three proteins are identical.

Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo.

A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera. Restriction enzyme analysis indicated that this DNA fragment was located on the B. burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A. Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichia coli chromosomal open reading frame.

Characterisation of Borrelia burgdorferi sensu lato strains isolated from patients with skin manifestations of Lyme borreliosis residing in Slovenia.

Lyme borreliosis is the most prevalent tick-borne infection in Slovenia. Skin disorders are the most frequent clinical manifestations. The aim of the present study was to assess the phenotypic and genotypic diversity of a large number of human Borrelia burgdorferi sensu lato isolates and to evaluate any association between the isolates and different clinical manifestations. All 103 strains tested were from patients suffering from the skin disorders of Lyme borreliosis. Skin biopsies, cerebrospinal fluid and blood samples from patients were inoculated into modified Kelly Pettenkofer medium. Protein profiles were determined by SDS-PAGE and species identification and plasmid profiles by pulsed-field gel electrophoresis. MluI digestion profiles showed that 87 (84.5%) isolates belonged to B. afzelii, 15 (14.5%) to B. garinii and 1 (1%) to B. burgdorferi sensu stricto. The number of plasmids in each strain varied from three to seven, and the plasmid size ranged from 15 to 65 kb. Four isolates of B. garinii possessed multiple large plasmids and four isolates had a large plasmid dimer (three B. afzelii and one B. garinii). Isolates showed qualitative and quantitative differences in protein expression. The study found differences in the expression of OspB and OspC proteins between B. afzelii and B. garinii strains. OspB was expressed significantly more often by B. afzelii (78 of 87, 89.6%) than by B. garinii (4 of 15, 26.6%) isolates, while OspC protein was expressed significantly more often by B. garinii (14 of 15, 93.3%) than by B. afzelii (51 of 87, 58.6%) isolates. In Slovenia, B. afzelii causes the majority of skin lesions. The isolates investigated showed plasmid and protein diversity. Heterogeneity of the spirochaetes may be important for virulence, and may have implications for pathogenesis and therapy of the infection. Differences in immunodominant proteins also have an important impact on serological testing and vaccine development.