Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 A. OspC, a largely alpha-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long alpha-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.     

Relationship between infectivity and OspC expression in Lyme disease Borrelia

Abstract Borrelia burgdorferi sensu stricto strain 297 and B. garinii strains HP1 and 12–92 were serially subcultured for 36–50 passages in vitro for 1 year. All low-passage strains showed abundant expression of outer surface protein C (OspC) in the 22–23-kDa range, but the high-passage strains lost or showed reduced expression of OspC in comparison with the low-passage strains. The low-passage strains efficiently infected outbred ddY mice when inoculated into the hind footpad or peritoneal cavity. In contrast, the incidence of infection with the high-passage strains was low. Isolates from the bladders of mice inoculated with the high-passage strains expressed large amounts of OspC in comparison with those originally inoculated. These results indicate that OspC expression is related to the infectivity of Lyme disease borreliae.     

Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.     

Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: A Study of OspC Proteins of Borrelia burgdorferi

Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants.     

Characterization of a unique borreliacidal epitope on the outer surface protein C of Borrelia burgdorferi

The outer surface protein C (OspC) of the Lyme disease agent, Borrelia burgdorferi, is an immunoprotective antigen in laboratory models of infection. However, to understand its protective effects, it is important to identify the key epitopes of this protein. We produced a borreliacidal anti-OspC monoclonal antibody specific to the B31 strain and identified its binding site. The specificity of MAb 16.22 was determined by Western blot reactivity using OspC derived from different Borrelia isolates which had varying amino acid sequences. Comparison of the OspC sequences and binding data suggested that MAb 16.22 binds to amino acids 133-147 of the OspC protein. To test this hypothesis, we synthesized a 15-amino acid peptide containing the target sequence and, using competition enzyme-linked immunosorbent assay (ELISA), we found that this peptide included the epitope of MAb 16.22. In addition, we determined that MAb 16.22 is able to kill of B. burgdorferi in a complement-independent fashion.     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

Hepatitis B virus capsid-like particles can display the complete, dimeric outer surface protein C and stimulate production of protective antibody responses against Borrelia burgdorferi infection

Hepatitis B virus capsid-like particles (CLPs), icosahedral assemblies formed by 90 or 120 core protein dimers, hold promise as immune-enhancing vaccine carriers for heterologous antigens. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop, are especially immunogenic. However, display of whole proteins, desirable to induce multispecific and possibly neutralizing antibody responses, can be restrained by an unsuitable structure of the foreign protein and by its propensity to undergo homomeric interactions. Here we analyzed CLP formation by core fusions with two distinct variants of the dimeric outer surface lipoprotein C (OspC) of the Lyme disease agent Borrelia burgdorferi. Although the topology of the termini in the OspC dimer does not match that of the insertion sites in the carrier dimer, both fusions, coreOspCa and coreOspCb, efficiently formed stable CLPs. Electron cryomicroscopy clearly revealed the surface disposition of the OspC domains, possibly with OspC dimerization occurring across different core protein dimers. In mice, both CLP preparations induced high-titered antibody responses against the homologous OspC variant, but with substantial cross-reactivity against the other variant. Importantly, both conferred protection to mice challenged with B. burgdorferi. These data show the principal applicability of hepatitis B virus CLPs for the display of dimeric proteins, demonstrate the presence in OspC of hitherto uncharacterized epitopes, and suggest that OspC, despite its genetic variability, may be a valid vaccine candidate.     

Identification of residues within ligand‐binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment

Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1′ of the apposing monomers form a solvent‐accessible pocket at the dimeric interface that presents a putative ligand‐binding domain (LBD1). Here we employ site‐directed and allelic‐exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure–functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.     

Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Isolation of the recombinant proteins OspC and the fragment FlaA (F-FLAA) from the western-Siberian Borrelia garinii NT29 isolates and the study of their immunochemical properties

The structural proteins OspC and FlaA of the Lyme disease (LD) agent are known to be the basic antigens, which induce the humoral immune response at the initial stage of the disease. The goal of this work was to obtain the recombinant OspC and a fragment of the FlaA protein (f-FlaA) from the Western Siberian Borrelia garinii NT29 isolates and to assess the possibility of their use for the LD diagnosis. Encoding regions of the OspC and f-FlaB genes were amplified using PCR inserted in the pREB expressive vectors and cloned in the E. coli str. BL21 and C-600, respectively. The recombinant OspC and f-FlaA proteins were purified using affinity chromatography on Ni-NTA-sepharose 6A, and their ability to bind serum antibody of patients with Lyme disease was tested using western-blot and ELISA methods. The results of the analyses suggest that these proteins can be considered as promising components for elaboration of diagnostic tests for LD. The prototype of the ELISA diagnostic test was designed on the basis of the OspC and f-FlaA recombinant antigens. This test provides satisfactory parameters of diagnostic specificity (70.0%) and sensitivity (85.0%).     

Isolation and purification of recombinant outer surface protein C (rOspC) of Borrelia burgdorferi sensu lato

The aim of this work was isolation and purification of the major immunodominant protein, Outer surface protein C (OspC) of three members of the species group Borrelia burgdorferi, the causative agent of Lyme disease. Our aim was to obtain this protein in a quantity and purity sufficient for immunization of experimental animals. For optimalization of protein purification's yield we used immobilized metal ion affinity chromatography (IMAC) under different conditions. The greatest efficiency was achieved by using of HiTrap Chelating Column under native conditions.     

Lipoprotein succession in Borrelia burgdorferi: similar but distinct roles for OspC and VlsE at different stages of mammalian infection

Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include shifting among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that these proteins carry out a basic but essential function, and that OspC and VlsE fulfil this requirement during early and persistent stages of mammalian infection respectively. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection. In contrast, we determined that OspC production was required by B. burgdorferi throughout SCID mouse infection if the vlsE gene were absent. Together, these results indicate that OspC can substitute for VlsE when antigenic variation is unnecessary, but that these two abundant lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi.     

Maximum antigen diversification in a lyme bacterial population and evolutionary strategies to overcome pathogen diversity

Natural populations of pathogens and their hosts are engaged in an arms race in which the pathogens diversify to escape host immunity while the hosts evolve novel immunity. This co-evolutionary process poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show high antigenic specificities of natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-informed strategies to broaden OspC antigenic reactivity. In particular, the centroid algorithm—a genetic algorithm to generate sequences that minimize amino-acid differences with natural variants—generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains co-existing in the Northeast United States. Mechanistically, we propose a model of maximum antigen diversification (MAD) mediated by amino-acid variations distributed across the hypervariable regions on the OspC molecule. Under the MAD hypothesis, evolutionary centroids display broad cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to vaccine designs based on concatenated epitopes, the evolutionary algorithms generate analogs of natural antigens and are automated. The novel centroid algorithm and the evolutionary antigen designs based on consensus and ancestral sequences have broad implications for combating diversifying pathogens driven by pathogen–host co-evolution.Subject terms: Population genetics, Bacterial genetics     

Changes in Bacterial Growth Rate Govern Expression of the Borrelia burgdorferi OspC and Erp Infection-Associated Surface Proteins

The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that the synthesis of OspC and Erps can be stimulated in culture by shifting the temperature from 23°C to 34°C, leading to a hypothesis that Borrelia burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing a separate evaluation of each factor''s impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase the expression of the OspC and Erp infection-associated proteins.     

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.