Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi

Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the tick, but the clone lacking lp36 demonstrated low infectivity in the mammal. Restoration of lp36 to the mutant strain confirmed that the infectivity defect was due to loss of lp36. Moreover, spirochetes lacking lp36 exhibited a nearly 4-log increase in ID(50) relative to the isogenic lp36(+) clone. The infectivity defect of lp36-minus spirochetes was localized, in part, to loss of the bbk17 (adeC) gene, which encodes an adenine deaminase. This work establishes a vital role for lp36 in the infectious cycle of B. burgdorferi and identifies the bbk17 gene as a component of this plasmid that contributes to mammalian infectivity.     

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.     

Use of an Endogenous Plasmid Locus for Stable in trans Complementation in Borrelia burgdorferi

Targeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirochete Borrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers than B. burgdorferi plasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle. B. burgdorferi has over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochete in vivo but relatively unstable during in vitro cultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number and in vivo stability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into the bbe02 locus, a site on lp25 that was previously shown to be nonessential during both in vitro and in vivo growth. We demonstrate the functional utility of this strategy by restoring infectivity to an ospC mutant through complementation at this site on lp25 and stable maintenance of the ospC gene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation in B. burgdorferi.     

Borrelia burgdorferi small lipoprotein Lp6.6 is a member of multiple protein complexes in the outer membrane and facilitates pathogen transmission from ticks to mice

Borrelia burgdorferi lipoprotein Lp6.6 is a differentially produced spirochete antigen. An assessment of lp6.6 expression covering representative stages of the infectious cycle of spirochetes demonstrates that the gene is solely expressed during pathogen persistence in ticks. Deletion of lp6.6 in infectious B. burgdorferi did not influence in vitro growth, or its ability to persist and induce inflammation in mice, migrate to larval or nymphal ticks or survive through the larval-nymphal molt. However, Lp6.6-deficient spirochetes displayed significant impairment in their ability to transmit from infected ticks to naïve mice, which was restored upon genetic complementation of the mutant with a wild-type copy of lp6.6 , establishing that Lp6.6 plays a role in pathogen transmission from ticks to mammals. Lp6.6 is a subsurface, yet highly abundant, outer membrane antigen. Two-dimensional blue native/SDS-PAGE coupled with liquid chromatography-mass spectrometry (LC-MS/MS) analysis and protein cross-linking studies independently shows that Lp6.6 exists in multiple protein complexes in the outer membrane. We speculate that the function of Lp6.6 is connected to the physiological processes of these membrane complexes. Further characterization of differentially produced membrane antigens and associated protein complexes will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.     

Mapping of essential replication functions of the linear plasmid lp17 of B. burgdorferi by targeted deletion walking

The genome of the Lyme disease pathogen Borrelia burgdorferi strain B31 MI includes one linear chromosome, 10 circular and 12 linear plasmids. Members of four paralogous gene families, revealed by genome sequencing, have been suggested as replication/partition functions for both the linear and circular plasmids. Some of these genes have been experimentally shown to be essential for the replication of the B. burgdorferi replicons that encode them. In this study, we located the region essential for replication of lp17, the second smallest linear plasmid in B. burgdorferi. We used a novel in vivo method, targeted deletion walking, to systematically delete DNA from either the left or right end of lp17. We report that the region essential for replication of lp17 is 1.8 kb (bp 7946-9766) and contains only one intact open reading frame (BBD14). Expression of BBD14 is required for the replication, suggesting that it is the replication initiator for lp17. The BBD14 protein is a member of paralogous family (PF) 62 and we present the first experimental evidence for the role of a PF 62 member. Adjacent non-coding sequences are also required, suggesting that the origin lies at least partially outside the coding region. Surprisingly, deletion of BBD21, the ParA orthologue (PF 32), had little effect upon plasmid stability or incompatibility. Finally, data are presented suggesting that lp17 replication occurs preferentially on a linear rather than a circular DNA molecule.     

High-throughput plasmid content analysis of Borrelia burgdorferi B31 by using Luminex multiplex technology

Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain B. burgdorferi B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many B. burgdorferi strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage B. burgdorferi B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease Borrelia studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease Borrelia strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.     

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.     

Molecular interactions that enable movement of the Lyme disease agent from the tick gut into the hemolymph

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)(2) fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.     

CSM murray award lecture - functional studies of the Lyme disease spirochete - from molecules to mice

Lyme borreliosis, also known as Lyme disease, is now the most common vector transmitted disease in the northern hemisphere. It is caused by the spirochete Borrelia burgdorferi and related species. In addition to their clinical importance, these organisms are fascinating to study because of the wide variety of unusual features they possess. Ongoing work in the laboratory in several areas will be described. (1) The segmented genomes contain up to two dozen genetic elements, the majority of which are linear with covalently closed hairpin ends. These linear DNAs also display a very high degree of ongoing genetic rearrangement. Mechanisms for these processes will be described. (2) Persistent infection by Borrelia species requires antigenic variation through a complex DNA rearrangement process at the vlsE locus on the linear plasmid lp28-1. Novel features of this recombination process will be presented. (3) Evidence for a new global regulatory pathway of B. burgdorferi gene expression that is required for pathogenicity will be described. The DEAH box RNA helicase HrpA is involved in this pathway, which may be relevant in other bacteria. (4) The mechanism of B. burgdorferi to effectively disseminate throughout its host is being studied in real time by high resolution intravital imaging in live mice. Recent work will be presented.     

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.     

In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56

Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies—so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan^r) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan^r mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.     

The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens

The linear plasmid, lp28-1, is required for persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. This plasmid contains the vls antigenic variation locus, which has long been thought to be important for immune evasion. However, the role of the vls locus as a virulence factor during mammalian infection has not been clearly defined. We report the successful removal of the vls locus through telomere resolvase-mediated targeted deletion, and demonstrate the absolute requirement of this lp28-1 component for persistence in the mouse host. Moreover, successful infection of C3H/HeN mice with an lp28-1 plasmid in which the left portion was deleted excludes participation of other lp28-1 non-vls genes in spirochete virulence, persistence and the process of recombinational switching at vlsE. Data are also presented that cast doubt on an immune evasion mechanism whereby VlsE directly masks other surface antigens similar to what has been observed for several other pathogens that undergo recombinational antigenic variation.     

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.     

Telomere exchange between linear replicons of Borrelia burgdorferi

Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres. The overall organization of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes. We present evidence here that a rather recent nonhomologous recombination event in the B. burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which is present in the strain B31 lineage. At least two successive rounds of addition of linear plasmid genetic material to the chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism by which plasmid genetic material can become part of the chromosome. The unusual nonhomologous nature of this rearrangement suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B. burgdorferi chromosomes.     

Molecular characterization of Lactobacillus casei strains

Abstract The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.     

Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ～900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.