Genetic diversity and the absence of regional differences of Borrelia garinii as demonstrated by ospA and ospB gene sequence analysis

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR-RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6-kb DNA fragment (0.7-kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR-RFLP groups and two independent borrelial strains. The representatives in each PCR-RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor-joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR-RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.     

Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.     

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA

The ospA gene of Borrelia burgdorferi encodes an outer membrane protein which is a major antigen of the Lyme disease agent. Two sequence-specific sets of oligonucleotide primers were used to specify the amplification of the ospA coding sequence by the polymerase chain reaction. One set allowed the entire ospA sequence to be amplified, while the other primed amplification of a truncated form of ospA lacking the first 17 codons specified by the wild-type ospA structural gene, residues believed to constitute a signal sequence which normally would direct localization of the ospA protein to the Borrelia cell's outer membrane. Each set of primers also contained sequences near their 5' ends which facilitated cloning of the amplified DNA directly into a high level expression system based on bacteriophage T7 genetic elements. We showed that the full-length OspA protein is synthesized poorly in Escherichia coli and it is associated with the insoluble membrane fraction. In contrast, the truncated form can be expressed to very high levels and it is soluble. The truncated protein was purified to homogeneity and partially characterized. Its N-terminal sequence and molecular weight derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis agree with those deduced from the DNA sequence. It is a monomer with a native molecular weight of 28,000 and it is very resistant to digestion by trypsin even though it is rather rich in lysine residues (16 mol%). Recombinant OspA protein synthesized in E. coli is recognized by antibodies in sera of Lyme patients, which suggests that the protein may be useful in immunoassays and as a possible immunogen to protect against Lyme borreliosis.     

Sequence of the complete osp operon encoding two major outer membrane proteins of a European Borrelia burgdorferi isolate (B29)

The nucleotide sequence of the operon encoding the major outer surface proteins, OspA and OspB, of a European isolate of Borrelia burgdorferi (strain B29) was determined and compared to the osp operon of the American strain, B31. An amino acid (aa) identity of 80.7% was found when comparing the OspA of B29 with that of B31, whereas the aa sequence of the OspB of B29 reveals only 61.3% identity with the OspB of B31. Thus, strains B31 and B29 can be regarded as representatives of different B. burgdorferi groups.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

Phenotypic and genotypic analysis of<Emphasis Type="Italic">Borrelia</Emphasis> spp. isolated from<Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks by using electrophoretic chips and real-time polymerase chain reaction

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.     

The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms.

The human immune response to natural infection with Borrelia burgdorferi appears to differ from that seen in small mammals infected by needle inoculation. In humans, antibody to outer surface proteins A and B (OspA and OspB) is not detectable until late in infection, but small mammals inoculated with B. burgdorferi produce early antibody to OspA and OspB. To investigate this disparity we compared the immune response in hamsters to B. burgdorferi after needle inoculation with cultured organisms or infected tick homogenates with the immune response after tick transmitted (natural) infection. We determined that the antibody response to OspA and OspB after natural infection of hamsters is similar to that seen in humans, and differs from the antibody response after hamster infection by needle inoculation. High titers of antibody to OspA and OspB were undetectable even 42 wk after bite by B. burgdorferi-infected ticks. The failure to produce antibody to OspA and OspB was not dependent on challenge dose, because animals inoculated by needle with low doses (1 x 10(5) to 1 x 10(6) cells) of B. burgdorferi produced antibody to OspA and OspB. A rapid but limited anti-41-kDa response was observed. One possible new Ag, 43 kDa (p43), was identified. The antibody response to p43 was independent of the route of inoculation. Our results suggest that the hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle inoculated, cultured organisms.     

Cross-antigenicity between the major surface proteins (ospA and ospB) and other proteins of Borrelia burgdorferi

Two of the major surface Ag of Borrelia burgdorferi, the 31-kDa OspA and 34-kDa OspB proteins, are encoded by a 49-kb plasmid. In this study, mAb and monospecific polyclonal antibodies were used to define cross-antigenicity of the OspA and OspB protein to each other and to other lower molecular mass proteins by Western blot analysis. Two mAb studied, 105.5 and 184.1, were directed predominantly against the 31-kDa OspA protein. However, each also reacted with other minor bands, though with different specificities. Using V8 protease digestion and cleavage by cyanogen bromide, we demonstrated that each mAb reacted to the 31-kDa protein differently. Monospecific polyclonal rabbit and human antibodies directed against the 34-, 31-, 22-, and 20-kDa proteins were eluted from blots and used to further corroborate the cross-reactivity among these Ag. Rabbit antibodies to the 31- and 22-kDa Ag gave remarkably similar peptide maps after V8 protease digestion of the 31-kDa OspA protein, as did mAb 184.1, suggesting that this mAb recognized an immunodominant epitope common to the 22- and 31-kDa proteins. It seems likely therefore that the humoral immune response to Borrelia surface Ag may be due to a limited number of cross-reactive epitopes on distinct, but related, gene products.     

Deletion in the Genes Encoding Outer Surface Proteins OspA and OspB of Borrelia garinii Isolated from Patients in Japan

We detected the expression of outer surface proteins OspA and OspB, and characterized the genes encoding the two Osps of eight Borrelia garinii isolates from patients in Japan. Six of the eight strains shared a common antigenic epitope in their OspA and/or OspB proteins to monoclonal antibody P3134 against OspB, and were identified to have a conserved carboxyl terminus on their ospA and ospB genes by Southern blot hybridization. One strain, JEM4, did not express OspB protein, which was due to lack of the ospB gene. Gene cloning and sequencing analysis revealed that it had only one osp open reading frame with 819 nucleotides, which was similar to the ospA gene. The deletion of the ospB gene could be explained by a homologous recombination based on the common C-terminal sequences on the ospAB operon.     

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.     

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.     

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi

Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologous genes encoding the major outer surface proteins (Osps) A and B of B. burgdorferi which both deletes osp gene sequences and creates chimaeric gene fusions. Recombinant osp genes occur in multiple strains and encode unique proteins that lack some characteristic Osp epitopes. Antigenic variation in Osp through recombination may be relevant to the persistence of B. burgdorferi in an infected host, and has important implications for the utility of OspA and OspB as diagnostic or vaccine candidates for Lyme disease. We also describe Osp variation arising from nonsense mutations and sequence divergence, which may also represent significant sources of Osp polymorphism.     

Detection of a large linear plasmid in Borrelia spielmanii isolate

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.     

Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene

The nucleotide sequence of the Escherichia coli colicin I receptor gene (cir) has been determined. The predicted mature protein consists of 599 amino acids and has a molecular weight of 67,169. Several previously noted characteristics of other E. coli outer membrane protein sequences were also identified in the sequence of Cir. These include an overall acidic nature, the absence of long hydrophobic stretches of amino acids, and a lack of predicted alpha-helical secondary structure. Because two classes of outer membrane proteins (the TonB-dependent transport proteins and the porins) share some structural features, protein sequences from both of these groups were aligned pairwise and scored for sequence similarity. Statistical evidence suggested that the porins were not related to the proteins in the TonB-dependent group; however, there was a significant relationship between the proteins in the TonB-dependent group. On the basis of the multiple progressive sequence alignment and the similarity scores derived from it, a tree representing evolutionary distance between five TonB-dependent outer membrane transport proteins was generated.     

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.     

Characterization of Borrelia garinii isolated from Lyme disease patients in Hokkaido, Japan, by sequence analysis of OspA and OspB genes

The outer surface proteins OspA and OspB genes of clinical Borrelia garinii isolates (JEM1–8) from Hokkaido, Japan were sequenced. One strain, JEM4, has a single ospA gene which is similar to European B. garinii strains PBr (sequence homology value: 94.1%) and T25 (91.2%). Five of the other seven strains exhibit a homologous C-terminus (300 bp) on both ospA and ospB genes (88.7–97.3%). The other two strains seem to be derived from the five strains by ospA or ospB alterations. In a phylogenetic analysis based on ospA, these strains could be classified into B. garinii, but formed independent branches and separated from the typical B. garinii isolates from Europe and Russia.     

Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.