Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi

The ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi. The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29,334, and (OspB) 296 amino acids long with a molecular weight of 31,739. The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event. Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins. These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines.     

OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis

No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).     

Molecular diagnosis of Borrelia burgdorferi infection (Lyme disease).

In spite of significant advances in immunologically based testing, accurate diagnosis of Lyme borreliosis remains problematic. To address this issue, a DNA amplification-based diagnostic test was developed utilizing the polymerase chain reaction (PCR) and oligonucleotide primers specific for the OspA and OspB genes of Borrelia burgdorferi. In this approach, a relatively large DNA fragment is amplified with an outer set of primers, and a "nested" internal sequence of the PCR product subsequently reamplified with an inner set of primers. This nested approach coupled with simple differential centrifugation allowed specific detection of as few as four B. burgdorferi organisms mixed in 2 ml of blood. This methodology was utilized on patients' samples, and it allowed detection of B. burgdorferi in the peripheral blood and urine of several individuals with clinical evidence of Lyme borreliosis. PCR became negative and symptoms improved following antibiotic therapy of treated individuals. These studies suggest that direct detection of Borrelia in infected individuals can aid in diagnosis and evaluation of therapy for Lyme borreliosis.     

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli

Abstract Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi . This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD . The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains ix histidyl residues in its N-terminus.     

Structural and molecular analysis of a protective epitope of Lyme disease antigen OspA and antibody interactions

The murine monoclonal antibody LA‐2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi , the causative agent of Lyme disease in North America. Human antibody equivalence to LA‐2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA‐2 protective epitope is important for developing OspA‐based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure‐based analysis of the LA‐2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA‐2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA‐2 light chain, N52 on the LA‐2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA‐2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.     

Translocation of Borrelia burgdorferi surface lipoprotein OspA through the outer membrane requires an unfolded conformation and can initiate at the C‐terminus

Borrelia burgdorferi surface lipoproteins are essential to the pathogenesis of Lyme borreliosis, but the mechanisms responsible for their localization are only beginning to emerge. We have previously demonstrated the critical nature of the amino‐terminal ‘tether’ domain of the mature lipoprotein for sorting a fluorescent reporter to the Borrelia cell surface. Here, we show that individual deletion of four contiguous residues within the tether of major surface lipoprotein OspA results in its inefficient translocation across the Borrelia outer membrane. Intriguingly, C‐terminal epitope tags of these N‐terminal deletion mutants were selectively surface‐exposed. Fold‐destabilizing C‐terminal point mutations and deletions did not block OspA secretion, but rather restored one of the otherwise periplasmic tether mutants to the bacterial surface. Together, these data indicate that disturbance of a confined tether feature leads to premature folding of OspA in the periplasm and thereby prevents secretion through the outer membrane. Furthermore, they suggest that OspA emerges tail‐first on the bacterial surface, yet independent of a specific C‐terminal targeting peptide sequence.     

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.     

Unfolding mechanics of multiple OspA substructures investigated with single molecule force spectroscopy

We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.     

Epitope mapping of the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi using a panel of monoclonal antibodies and lanthanide competition fluoroimmunoassay

A panel of fourteen different monoclonal antibodies was used for detection and analysis of antigenic determinants located on the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi, which is a causative agent of tick-borne borreliosis (Lyme disease). Two main and several minor partially overlapping antigenic determinants have been found on the surface of the OspA protein of Borrelia burgdorferi sensu stricto (strain 297) by lanthanide competition fluoroimmunoassay. One of the main antigenic determinants is located in the N- and the other in the C-half of the OspA molecule. The involvement of the OspA protein in intact Borrelia burgdorferi sensu stricto (four bacterial strains have been analyzed: 297, B31, FR90-594, and CA90-742) is associated with retention of the above-mentioned two major antigenic determinants, but unlike the case of the isolated OspA they are partially overlapping with each other and with other antigenic determinants. The protein of the spirochete Borrelia afzelii (two bacterial strains have been analyzed: Ip-21 and Pko) contains only one antigenic determinant, which is the same as the main determinant of the OspA protein of Borrelia burgdorferi sensu stricto located in the N-half of the OspA molecule.     

Structure-based design of a second-generation Lyme disease vaccine based on a C-terminal fragment of Borrelia burgdorferi OspA

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi elicits protective immunity that blocks transmission of Borrelia from the tick vector to the vaccinated animal, and thus has been a focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer beta-sheet. All anti-OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of using the C-terminal domain alone as a recombinant protein-based vaccine. The removal of ineffective parts from the OspA antigen may reduce side effects and lead to a safer vaccine. We prepared a C-terminal fragment of OspA by removing approximately 45% of residues from the N terminus. Although the fragment retained the native conformation and affinity to a protective antibody, its vaccine efficacy and conformational stability were significantly reduced with respect to full-length OspA. We successfully stabilized the fragment by replacing amino acid residues involved in buried salt-bridges with residues promoting hydrophobic interactions. The mutations promoted the vaccine efficacy of the redesigned fragment to a level comparable to that of the full-length protein, demonstrating the importance of the antigen stability for OspA's vaccine efficacy. Our strategy should be useful for further refining OspA-based vaccines and developing recombinant vaccines for other diseases.     

Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease.

We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.     

环介导恒温扩增对莱姆病病原伯氏疏螺旋体的分型鉴定

使用环介导恒温扩增技术,基于莱姆病病原伯氏疏螺旋体的外膜蛋白A(OspA)基因,针对伯氏疏螺旋体不同的基因型设计特异性引物,对国内主要的莱姆病病原伯氏疏螺旋体的3个基因型进行分型鉴定。研究结果表明,设计的引物具有良好的特异性,可以对狭义伯氏疏螺旋体(Borrelia burgdorferi sensu strict)、嘎氏疏螺旋体(B.afzelii)和伽氏疏螺旋体(B.garinii)进行分型鉴定。伯氏疏螺旋体的分型鉴定可以对不同临床症状莱姆病患者的治疗和莱姆病的控制提供一定的依据。     

Probing the Borrelia burgdorferi surface lipoprotein secretion pathway using a conditionally folding protein domain

Surface lipoproteins of Borrelia spirochetes are important virulence determinants in the transmission and pathogenesis of Lyme disease and relapsing fever. To further define the conformational secretion requirements and to identify potential lipoprotein translocation intermediates associated with the bacterial outer membrane (OM), we generated constructs in which Borrelia burgdorferi outer surface lipoprotein A (OspA) was fused to calmodulin (CaM), a conserved eukaryotic protein undergoing calcium-dependent folding. Protein localization assays showed that constructs in which CaM was fused to full-length wild-type (wt) OspA or to an intact OspA N-terminal "tether" peptide retained their competence for OM translocation even in the presence of calcium. In contrast, constructs in which CaM was fused to truncated or mutant OspA N-terminal tether peptides were targeted to the periplasmic leaflet of the OM in the presence of calcium but could be flipped to the bacterial surface upon calcium chelation. This indicated that in the absence of an intact tether peptide, unfolding of the CaM moiety was required in order to facilitate OM traversal. Together, these data further support a periplasmic tether peptide-mediated mechanism to prevent premature folding of B. burgdorferi surface lipoproteins. The specific shift in the OM topology of sequence-identical lipopeptides due to a single-variable change in environmental conditions also indicates that surface-bound Borrelia lipoproteins can localize transiently to the periplasmic leaflet of the OM.     

Borrelia burgdorferi lipoproteins are secreted to the outer surface by default

Borrelia spirochaetes are unique among diderm bacteria in their abundance of surface-displayed lipoproteins, some of which play important roles in the pathogenesis of Lyme disease and relapsing fever. To identify the lipoprotein-sorting signals in Borrelia burgdorferi, we generated chimeras between the outer surface lipoprotein OspA, the periplasmic oligopeptide-binding lipoprotein OppAIV and mRFP1, a monomeric red fluorescent reporter protein. Localization of OspA and OppAIV point mutants showed that Borrelia lipoproteins do not follow the '+2' sorting rule which targets lipoproteins to the cytoplasmic or outer membrane of Gram-negative bacteria via the Lol pathway. Fusions of mRFP1 to short N-terminal lipopeptides of OspA, and surprisingly OppAIV, were targeted to the spirochaetal surface. Mutagenesis of the OspA N-terminus defined less than five N-terminal amino acids as the minimal secretion-facilitating signal. With the exception of negative charges, which can act as partial subsurface retention signals in certain peptide contexts, lipoprotein secretion occurs independent of N-terminal sequence. Together, these data indicate that Borrelia lipoproteins are targeted to the bacterial surface by default, but can be retained in the periplasm by sequence-specific signals.     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

Expression and Secretion of Recombinant Outer-surface Protein A from the Lyme Disease Agent, Borrelia burgdorferi, in Nicotiana tabacum Suspension Cells

The ospA gene of Borrelia burgdorferi codes for an outer membrane lipoprotein, which is a major antigen of the Lyme disease agent. Recombinant OspA vaccines tested so far were expressed in Escherichia coli. In this study, we investigated the expression of a soluble OspA protein in Nicotiana tabacum suspension cells and evaluated the secretion of OspA driven by either its own bacterial signal peptide or a plant signal peptide fused to the amino-terminal cysteine of the mature form. In both cases, the signal peptide was cleaved off and OspA secreted. During secretion, OspA was N-glycosylated. Addition of a C-terminal KDEL sequence led to retention of OspA in the endoplasmic reticulum.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

Production of outer surface protein A by Borrelia burgdorferi during transmission from infected mammals to feeding ticks is insufficient to trigger OspA seroconversion

The Lyme disease spirochete, Borrelia burgdorferi, produces two outer surface lipoproteins, OspA and OspB, that are essential for colonization of tick vectors. Both proteins are highly expressed during transmission from infected mammals to feeding ticks and during colonization of tick midguts, but are repressed when bacteria are transmitted from ticks to mammals. Humans and other infected mammals generally do not produce antibodies against either protein, although some Lyme disease patients do seroconvert and produce antibodies against OspA for unknown reasons. We hypothesized that, if such patients had been fed upon by additional ticks, bacteria moving from the patients' bodies to the feeding ticks would have produced OspA and OspB proteins, which then led to immune system recognition and antibody production. This hypothesis was tested by analyzing immune responses of infected mice following feedings by additional Ixodes scapularis ticks. However, results of the present studies demonstrate that expression of OspA and OspB by B. burgdorferi during transmission from infected mammals to feeding ticks does not trigger seroconversion.