Distribution of twelve linear extrachromosomal DNAs in natural isolates of Lyme disease spirochetes

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts.     

Defining the plasmid-borne restriction-modification systems of the Lyme disease spirochete Borrelia burgdorferi

The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirochete Borrelia burgdorferi has two plasmid-borne putative R-M genes, bbe02 and bbq67, whose presence limits transformation by shuttle vector DNA from Escherichia coli. We show that both the bbe02 and bbq67 loci in recipient B. burgdorferi limit transformation with shuttle vector DNA from E. coli, irrespective of its dam, dcm, or hsd methylation status. However, plasmid DNA purified from B. burgdorferi transformed naïve B. burgdorferi much more efficiently than plasmid DNA from E. coli, particularly when the bbe02 and bbq67 genotypes of the B. burgdorferi DNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared from B. burgdorferi that carried bbe02 and bbq67. These results indicate that the bbe02 and bbq67 loci of B. burgdorferi encode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.Borrelia burgdorferi sensu lato is a zoonotic pathogen whose natural infectious cycle alternates between a tick vector and rodent or bird reservoir hosts (1, 7, 8, 14, 32, 33, 36). Transmission of B. burgdorferi to humans occurs through the bite of an infected tick and can lead to Lyme disease, which is a major public health concern in areas of North America and Europe where B. burgdorferi is endemic (8, 53).The genomic structure of the spirochete B. burgdorferi is unique, consisting of a linear chromosome of approximately 900 kb and more than 20 linear (lp) and circular (cp) plasmids, ranging in size from ∼5 kb to 56 kb, in the type strain B31 (9, 10, 11, 19, 42). The plasmids of B. burgdorferi are present at unit copy number relative to the chromosome (22), and some are relatively unstable during in vitro propagation (52, 57). The loss of linear plasmids lp25, lp28-1, and lp36 by strain B31 was found to correlate with the loss of infectivity in mice (20, 31, 45, 56), leading to the identification of genes carried on these plasmids that are dispensable in vitro but required in vivo during an experimental infectious cycle (21, 26, 35, 44, 47). The loss of two linear plasmids, lp25 and lp56, was shown to correlate with enhanced shuttle vector transformation, suggesting that specific lp25 and lp56 gene products present a barrier to stable introduction of foreign DNA (34). Further studies linked the transformation phenotype of B. burgdorferi strain B31 with the bbe02 and bbq67 genes on lp25 and lp56, respectively, and the putative restriction-modification (R-M) enzymes that they encode (11, 27, 29, 34). The recent demonstration by Chen and colleagues of enhanced transformation of B. burgdorferi following in vitro methylation of DNA (13) further supports the hypothesis that these B. burgdorferi plasmids encode R-M enzymes that degrade foreign DNA lacking the appropriate modification.The barrier to foreign DNA presented by the bbe02 and bbq67 loci of B. burgdorferi implies that genomic DNA should be modified in spirochetes carrying these plasmid genes. To test this hypothesis, we compared the transformation of B. burgdorferi with shuttle vector DNA isolated from either Escherichia coli or B. burgdorferi, as outlined in Fig. ​Fig.1.1. We also examined whether and how the presence of putative R-M genes in either the donor or recipient B. burgdorferi strain influenced transformation. Finally, we analyzed the type of modification present on DNA isolated from B. burgdorferi with different plasmid or gene contents. Our data indicate that the bbe02 and bbq67 loci of B. burgdorferi encode enzymes that both methylate endogenous DNA and restrict foreign DNA lacking these modifications. These findings have basic implications regarding horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. These results also help elucidate the molecular mechanisms underlying the relative inefficiency of genetic transformation of B. burgdorferi and suggest ways in which genetic manipulation of this pathogenic spirochete could be enhanced.Open in a separate windowFIG. 1.Shuttle vector transformations. Schematic representation of the various DNA sources, strains and methods used to assess the contributions of bbe02 and bbq67 to the restriction-modification (R-M) systems of B. burgdorferi.     

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi

Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues. GAG binding by B. burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (Borrelia GAG-binding protein), on the basis of its ability to bind to heparin and erythrocytes. Bgp was found in outer membrane fractions of B. burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane-impermeable biotinylating agent or anti-Bgp antibodies. Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B. burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B. burgdorferi binding to purified GAGs and to cultured mammalian cells. Thus, Bgp is a strong candidate for a GAG-binding adhesin of B. burgdorferi.     

The structure of a pyrophosphate-dependent phosphofructokinase from the Lyme disease spirochete Borrelia burgdorferi

The structure of the 60 kDa pyrophosphate (PP(i))-dependent phosphofructokinase (PFK) from Borrelia burgdorferi has been solved and refined (R(free) = 0.243) at 2.55 A resolution. The domain structure of eubacterial ATP-dependent PFKs is conserved in B. burgdorferi PFK, and there are three large insertions relative to E. coli PFK, including a helical domain containing a hairpin structure that interacts with the active site. Asp177, conserved in all PP(i) PFKs, negates the binding of the alpha-phosphate group of ATP and likely contacts the essential Mg(2+) cation via a water molecule. Asn181 blocks the binding of the adenine moiety of ATP. Lys203 hydrogen bonds to a sulfate anion that likely mimics PP(i) substrate binding.     

Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi

Hfq is a global regulatory RNA‐binding protein. We have identified and characterized an atypical Hfq required for gene regulation and infectivity in the Lyme disease spirochete Borrelia burgdorferi. Sequence analyses of the putative B. burgdorferi Hfq protein revealed only a modest level of similarity with the Hfq from Escherichia coli, although a few key residues are retained and the predicted tertiary structure is similar. Several lines of evidence suggest that the B. burgdorferi bb0268 gene encodes a functional Hfq homologue. First, the hfq_Bb gene (bb0268) restores the efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant. Second, the Hfq from B. burgdorferi binds to the small RNA DsrA_Bb and the rpoS mRNA. Third, a B. burgdorferi hfq null mutant was generated and has a pleiotropic phenotype that includes increased cell length and decreased growth rate, as found in hfq mutants in other bacteria. The hfq_Bb mutant phenotype is complemented in trans with the hfq gene from either B. burgdorferi or, surprisingly, E. coli. This is the first example of a heterologous bacterial gene complementing a B. burgdorferi mutant. The alternative sigma factor RpoS and the outer membrane lipoprotein OspC, which are induced by increased temperature and required for mammalian infection, are not upregulated in the hfq mutant. Consequently, the hfq mutant is not infectious by needle inoculation in the murine model. These data suggest that Hfq plays a key role in the regulation of pathogenicity factors in B. burgdorferi and we hypothesize that the spirochete has a complex Hfq‐dependent sRNA network.     

The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorferi

Seven hamsters, experimentally infected with Borrelia burgdorferi, were examined by both cultural and histological techniques at 1 to 9 months postinfection. Spirochetes were detected in the spleen, kidney, or eye of all animals by culture and in the spleen, kidney, eye, liver, or heart blood of five of seven animals by histological examination. Two animals showed nonspecific hepatic portal lymphocytic infiltration, while five of the hamsters displayed no significant histologic signs of inflammation or granuloma formation in the major organ systems. Synovitis and arthropathy did not occur. All animals showed some degree of follicular lymphoid hyperplasia of the spleen. Spirochetes were predominantly extracellular with a rare organism appearing to be partially within a macrophage.     

Natural exposure of Wisconsin dogs to the Lyme disease spirochete (Borrelia burgdorferi)

Lyme disease, a tick-borne disease caused by Borrelia burgdorferi, has been described recently in dogs. In a serologic survey of specimens obtained from March to September 1984, 53% of 380 dogs from two USDA licensed vendors in Wisconsin were positive for indirect immunofluorescent antibodies to B. burgdorferi at a serum dilution of 1:64 or greater. B. burgdorferi was cultured from the blood of 8/111 dogs. These findings suggest that exposure to this spirochete is common in endemic areas and that this zoonotic disease is of importance to veterinarians and researchers.     

Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy.

The etiological agent of Lyme disease is the tick-borne spirochete, Borrelia burgdorferi. A major antigen of B. burgdorferi is a 31 kDa lipoprotein called outer surface protein A (OspA). Recently, a truncated form of OspA (lacking 17 amino acids at the N-terminus) was cloned, expressed and purified in large quantities (Dunn, J.J., Lade, B.A. and Barbour, A.G. (1990) Protein Expression and Purification 1, 159-168). The truncated protein (OspA-257) is water-soluble, retains the ability to bind antibodies from the sera of Lyme disease patients and may prove useful in development of a vaccine against Lyme disease. We have used far UV circular dichroism (CD) and fluorescence spectroscopy to characterize the secondary structure of and to study conformational changes in OspA-257. CD spectra from 260 to 178 nm predict five classes of secondary structure: alpha-helix (11%), anti-parallel beta-sheet (32%), parallel beta-sheet (10%), beta-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for alpha-helical regions (residues 100-107, 121-134, 253-273) and for antigenic determinants (Lys-46, Asp-82, Lys-231). CD spectra of the native protein show little change from pH 3 to 11. Thermal denaturation curves, indicate that 'salt bridges' play a role in stabilizing the native protein. Both thermal and chemical denaturations that eliminate all secondary structure as judged by CD or fluorescence are reversible. Denaturation by guanidine-HCl (gdn-HCl) appears to be a cooperative, two-state transition, as indicated by a sudden change in the CD spectrum at approximately 0.75 M gdn-HCl, and an isodichroic point at 208 nm in all CD spectra measured from 0.0-1.75 M gdn-HCl.     

Incompetence of catbirds as reservoirs for the Lyme disease spirochete (Borrelia burgdorferi)

We compared the relative infectivity to vector ticks of gray catbirds (Dumetella carolinensis) and white-footed mice (Peromyscus leucopus) for the Lyme disease spirochete (Borrelia burgdorferi). Of 28 catbirds captured in a site enzootic for this agent, 18 were infested by immature Ixodes dammini, the tick vector. By comparison, each of 32 mice sampled concurrently from the same site was infested, and by about 10 times as many ticks as were found infesting the 3 most commonly netted bird species. Although 76% of noninfected larval ticks placed on these mice in a xenodiagnosis became infected, none of the ticks similarly placed on 12 catbirds did so. Spirochetes were detected in ticks derived from 2 Carolina wrens (Thryothorus ludovicianus) and a common yellowthroat (Geothlypis trichas), but these species' potential contribution to infecting ticks does not compare with that of mice. Thus, although birds may help establish new foci of ticks, catbirds, at least, do not appear to contribute as reservoirs of infection.     

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."     

Changes in antigenic reactivity of Borrelia burgdorferi, the Lyme disease spirochete, during persistent infection in mice

Adult laboratory mice, Mus musculus, were shown to be suitable experimental animals for studying infectivity, persistent infection, and in vivo antigenic changes of Borrelia burgdorferi. Sixteen mice were inoculated intraperitoneally with a low-passage culture of an uncloned strain of B. burgdorferi and 16 months later spirochetes were reisolated from the urinary bladder of 15 (94%) of the mice. Spirochetes recovered from the urinary bladder of one persistently infected mouse were tested for infectivity and found to be infectious when passaged into four laboratory mice. Western blot analysis of immune serum from each of the persistently infected mice demonstrated that spirochetes used to infect the mice reacted differently when compared with the spirochetes subsequently reisolated from the mice, demonstrating for the first time that changes in antigenic reactivity had occurred in the spirochete populations during persistent infection.     

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.     

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.     

Functional analysis of a lipid galactosyltransferase synthesizing the major envelope lipid in the Lyme disease spirochete Borrelia burgdorferi

One of the major lipids in the membranes of Borrelia burgdorferi is monogalactosyl diacylglycerol (MGalDAG), a glycolipid recently shown to carry antigenic potency. Herein, it is shown that the gene mgs (TIGR designation bb0454) of B. burgdorferi encodes for the protein bbMGS that, when expressed in Escherichia coli, catalyzes the glycosylation of 1,2-diacylglycerol with specificity for the donor substrate UDP-Gal yielding MGalDAG. Related lipid enzymes were found in many Gram-positive bacteria. The presence of this galactosyltransferase activity and synthesis of a cholesteryl galactoside by another enzyme were verified in B. burgdorferi cell extract. Besides MGalDAG, phosphatidylcholine, phosphatidylglycerol, and cholesterol were also found as major lipids in the cell envelope. The high isoelectric point of bbMGS and clustered basic residues in its amino acid sequence suggest that the enzyme interacts with acidic lipids in the plasma membrane, in agreement with strong enzymatic activation of bbMGS by phosphatidylglycerol. The membrane packing and immunological properties of MGalDAG are likely to be of great importance in vivo.     

Perpetuation of the Lyme disease spirochete Borrelia lusitaniae by lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.