Homology of variable major protein genes between Borrelia hermsii and Borrelia miyamotoi

Abstract Antigenic variation has been studied in detail for the etiological agent of relapsing fever, Borrelia hermsii . The variable major proteins (vmps) are found at its cell surface, enabling it to avoid the host's immune response. We have cloned and sequenced the vmp -gene ( vmp )-like sequences from the Borrelia miyamotoi strains HT31 and FR64b and the deduced amino acid sequences were compared with the published vmp proteins vmp3, vmp24, and vmp33 of B. hermsii . The sequences were aligned and revealed pairwise sequence identities ranging from 45 to 51%, and differences were scattered throughout the sequences. Southern hybridization using the cloned vmp -like sequence of strain HT31 as a probe suggested that the vmp homologues reside on the linear plasmids of B. miyamotoi . The probe hybridized weakly with B. hermsii linear plasmids and restriction digests. These results suggest that B. miyamotoi has sequences resembling the vmp genes in B. hermsii .     

New World relapsing fever Borrelia found in Ornithodoros porcinus ticks in central Tanzania

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene-based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty-one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick-borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick-borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.     

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.     

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.     

The presence of Borrelia valaisiana-related genospecies in ticks and a rodent in Taiwan

A field survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) in six counties of Taiwan. Spirochetes were successfully isolated from one rodent ear sample out of 485 rodent ears and 53 live, fed tick (Ixodes granulatus) samples. The spirochetes were confirmed to be B. burgdorferi s.l. by real-time PCR. In addition, 23 of 113 tick samples were tested positive for Borrelia DNA according to real-time PCR. The Borrelia isolate from the rodent and the 23 Borrelia DNA samples from the ticks were identified as B. valaisiana-related genospecies by phylogenetic analysis based on flagellin gene sequences. These findings suggest that the Borrelia valaisiana-related strains are maintained in a zoonotic cycle between tick vectors and reservoir hosts in Taiwan.     

Phylogeographic relationships of Ixodes uriae (Acari: Ixodidae) and their significance to transequatorial dispersal of Borrelia garinii

The seabird tick Ixodes uriae (Acari: Ixodidae) has a bi- and circumpolar distribution and is commonly infected with Lyme disease Borrelia. Identical Borrelia flagellin gene sequences have been detected in I. uriae from both the Northern and Southern Hemispheres, suggesting a transequatorial transport of Borrelia. Parsimony analysis of the internal transcribed spacer 2 (ITS2) and a part of 16S rDNA of I. uriae from the Northern and Southern Hemispheres indicated that northern and southern I. uriae might be reproductively separated. We hypothesize that Borrelia is probably not dispersed from one hemisphere to the other by ticks attached to seabirds.     

Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.     

Proteomic analysis of Lyme disease: global protein comparison of three strains of Borrelia burgdorferi

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing approximately 38% coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.     

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.     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

Molecular characterization of Borrelia isolates from ticks and mammals from the southern United States

Fifty Borrelia isolates from ticks and rodents from several geographic regions of the southern United States were analyzed by genomic macrorestriction analysis. Significant genetic diversity was observed among them. These isolates segregated into 4 major clusters and 10 subclusters, which are correlated with the genospecies distribution. Nineteen pulsed-field gel electrophoresis (PFGE) types were recognized among the isolates. The genospecies Borrelia andersonii and Borrelia bissettii consisted of 5 and 2 subclusters, respectively. Two subclusters comprised the Borrelia burgdorferi sensu stricto (s. s.) strains. These results indicated that PFGE is a suitable molecular typing method for B. burgdorferi at both the genospecies and strain levels. Seventeen representative isolates from different PFGE groups were analyzed by restriction fragment length polymorphism (RFLP) and sequence analysis of flaB. Twenty-three AluI, 3 CelII, and 11 DdeI RFLP patterns were found among strains from the B. burgdorferi sensu lato (s. l.) complex and the relapsing fever borreliae complex. Three genospecies in the B. burgdorferi s. l. complex and 1 species in the relapsing fever borreliae complex were recognized. Phylogenetic analysis based on nucleotide sequences of flaB indicated that all the Borrelia strains analyzed here could be divided into 2 parts, i.e., B. burgdorferi s. l. complex and the relapsing fever borreliae complex. The flaB appears to be a useful target gene to screen and identify strains from both B. burgdorferi s. l. and relapsing fever borreliae complexes.     

Borrelia tanukii Sp. Nov. and Borrelia turdae Sp. Nov. Found from Ixodid Ticks in Japan: Rapid Species Identification by 16S rRNA Gene-Targeted PCR Analysis

Based on the results of RFLP-ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species-specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.     

Genomic Characteristics of Chinese Borrelia burgdorferi Isolates

In China, B. burgdorferi, B.garinii, B. afzelii and B. yangtze sp. nov have been reported; B.garinii and B. afzelii are the main pathogenic genotypes. But until now only one Chinese strain was reported with whole genome sequence. In order to further understand the genomic characteristics and diversity of Chinese Borrelia strains, 5 isolates from China were sequenced and compared with the whole genome sequences of strains in other areas. The results showed a high degree of conservation within the linear chromosome of Chinese strains, whereas plasmid showed a much larger diversity according to the majority genomic information of plasmids. The genome sequences of the five Chinese strains were compared with the corresponding reference strains, respectively, according to the genospecies. Pairwise analysis demonstrates that there are only 70 SNPs between the genomes of CS4 and B31. However, there are many more SNPs between the genomes of QX-S13 and VS116, PD91 and PBi, FP1 and PKo, R9 and Pko, respectively. Gene comparison showed some important different genes. OspA was one of the important different genes. Comparative genomic studies have found that OspA gene sequences of PD91 and R9 had great differences compared with the sequence of B31. OspA gene sequence of R9 had a 96bp deletion; OspA gene of PD91 had two deletions: 9bp and 10 bp. To conclude, we showed the genomic characteristics of four genotype Chinese B. burgdorferi strains. The genomic sequence of B. yangtze sp. nov and differences from B. valaisiana were first reported. Comparative analysis of Chinese strains with the different Borrelia species from other areas will help us to understand evolution and pathogenesis of Chinese Borrelia burgdorferi strains.     

<Emphasis Type="Italic">Borrelia lusitaniae</Emphasis> OspA Gene Heterogeneity in Mediterranean Basin Area

In this study, Borrelia lusitaniae DNA extracted from ticks and lizards was used to amplify the outer surface protein A (OspA) gene in order to increase knowledge about sequence variability in the Mediterranean basin area, to better understand how Borrelia lusitaniae has evolved and how its distribution has expanded. Phylogenetic trees including Italian and reference sequences showed a clear separation of B. lusitaniae OspA strains in two different major clades. North African isolates form a clade with Portuguese POTIB strains, whereas Italian samples are grouped with German strains and a human Portuguese strain. This subdivision was supported by very high posterior probability values in the trees, by both analysis of molecular variance and selective pressure. These results, based on phylogenetic information contained in the OspA gene sequences, show the presence of two different B. lusitaniae strains circulating in the Mediterranean basin area, suggesting two different evolution paths.     

Borrelia burgdorferi strain 25015: characterization of outer surface protein A and vaccination against infection.

Mice vaccinated with outer surface protein A (OspA) from Borrelia burgdorferi strain N40 are protected from challenge with an intradermal syringe inoculum of B. burgdorferi strains N40, B31, and CD16. Vaccination experiments were done to determine if protection extended to strains 297 and 25015. We now show that OspA-N40 immunized mice are protected against challenge with strain 297, isolated from the cerebrospinal fluid of a patient with neuroborreliosis, but not against challenge with strain 25015, isolated from a tick in Millbrook, NY. The OspA gene from strain 25015 was therefore cloned and sequenced. The deduced OspA-25015 protein sequence differs from OspA-N40 at 40 of 273 amino acids. Furthermore, mice vaccinated with rOspA-25015 are protected from challenge with strain 25015 but not against strain N40. The results extend the usefulness of OspA as a vaccine candidate, but indicate that OspA can vary among strains of B. burgdorferi and that vaccination of mice with OspA-N40 does not protect against intradermal challenge with an inoculum of 10(4) strain 25015 spirochetes.     

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.     

Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii ) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).     

Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. l. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. l., and agreed well with the current classification of genospecies.     

Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.     

Methylated DNA in Borrelia species.

The DNA of Borrelia species was examined for the presence of methylated GATC sequences. The relapsing-fever Borrelia sp., B. coriaceae, and only 3 of 22 strains of B. burgdorferi contained adenine methylation systems. B. anserina lacked an adenine methylation system. Fundamental differences in DNA methylation exist among members of the genus Borrelia.