Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host.     

Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.     

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.     

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.     

Thermoregulation-dependent expression of Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12.

Resistance to the bactericidal action of normal human serum is one of the characteristics of virulent Yersinia enterocolitica. This property is attributable to the virulence plasmid harbored by pathogenic strains of the species. Serum resistance in Y. enterocolitica is thermoregulated, and its expression correlates well with the presence of virulence plasmid-encoded outer membrane proteins. To further examine the biochemical basis underlying resistance, we cloned a large segment (ca. 30 kilobases) of virulence plasmid DNA and studied the expression of plasmid-encoded outer membrane proteins in a serum-sensitive strain of Escherichia coli. The presence of the 160-kilodalton Y. enterocolitica-derived outer membrane protein 1 on E. coli transformants conferred a high degree of hydrophobicity, autoagglutinability, and resistance to serum killing. All of these properties were thermoregulated in E. coli with fidelity, suggesting that a functional thermoregulatory element was present in the cloned DNA. Elimination of protein 1 from the outer membrane of E. coli transformants by insertional inactivation of the structural gene with a Kanr gene cassette abrogated all of these properties and returned the serum-sensitive phenotype.     

aadA confers streptomycin resistance in Borrelia burgdorferi

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.     

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.     

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

Borrelia burgdorferi inhibits human neutrophil functions

Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.     

CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.     

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.     

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.     

The role of Borrelia burgdorferi outer surface proteins

Human pathogenic spirochetes causing Lyme disease belong to the Borrelia burgdorferi sensu lato complex. Borrelia burgdorferi organisms are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. These spirochetes are unique in that they can cause chronic infection and persist in the infected human, even though a robust humoral and cellular immune response is produced by the infected host. How this extracellular pathogen is able to evade the host immune response for such long periods of time is currently unclear. To gain a better understanding of how this organism persists in the infected human, many laboratories have focused on identifying and characterizing outer surface proteins of B.?burgdorferi. As the interface between B.?burgdorferi and its human host is its outer surface, proteins localized to the outer membrane must play an important role in dissemination, virulence, tissue tropism, and immune evasion. Over the last two decades, numerous outer surface proteins from B.?burgdorferi have been identified, and more recent studies have begun to elucidate the functional role(s) of many borrelial outer surface proteins. This review summarizes the outer surface proteins identified in B.?burgdorferi to date and provides detailed insight into the functions of many of these proteins as they relate to the unique parasitic strategy of this spirochetal pathogen.     

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.     

Borrelia burgdorferi downregulates ICAM-1 on human synovial cells in vitro.

Lyme arthritis following infection with Borrelia burgdorferi (B. burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown. To investigate how an infection with B. burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B. burgdorferi in vitro. Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis. It has been shown previously that B. burgdorferi can persist in the cytosol of human synovial cells. The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B. burgdorferi. A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2. To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed. After infection with B. burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer. Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1. Downregulation of ICAM-1 on synovial cells due to infection with B. burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.