An Inverted Repeat in the ospC Operator Is Required for Induction in Borrelia burgdorferi

Borrelia burgdorferi, the spirochete that causes Lyme disease, differentially regulates synthesis of the outer membrane lipoprotein OspC to infect its host. OspC is required to establish infection but then repressed in the mammal to avoid clearance by the adaptive immune response. Inverted repeats (IR) upstream of the promoter have been implicated as an operator to regulate ospC expression. We molecularly dissected the distal inverted repeat (dIR) of the ospC operator by site-directed mutagenesis at its endogenous location on the circular plasmid cp26. We found that disrupting the dIR but maintaining the proximal IR prevented induction of OspC synthesis by DNA supercoiling, temperature, and pH. Moreover, the base-pairing potential of the two halves of the dIR was more important than the nucleotide sequence in controlling OspC levels. These results describe a cis-acting element essential for the expression of the virulence factor OspC.     

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.     

Complete Genome Sequence of Borrelia afzelii K78 and Comparative Genome Analysis

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.     

Comparative analysis of the Borrelia garinii genome

Three members of the genus Borrelia (B.burgdorferi, B.garinii, B.afzelii) cause tick-borne borreliosis. Depending on the Borrelia species involved, the borreliosis differs in its clinical symptoms. Comparative genomics opens up a way to elucidate the underlying differences in Borrelia species. We analysed a low redundancy whole-genome shotgun (WGS) assembly of a B.garinii strain isolated from a patient with neuroborreliosis in comparison to the B.burgdorferi genome. This analysis reveals that most of the chromosome is conserved (92.7% identity on DNA as well as on amino acid level) in the two species, and no chromosomal rearrangement or larger insertions/deletions could be observed. Furthermore, two collinear plasmids (lp54 and cp26) seem to belong to the basic genome inventory of Borrelia species. These three collinear parts of the Borrelia genome encode 861 genes, which are orthologous in the two species examined. The majority of the genetic information of the other plasmids of B.burgdorferii is also present in B.garinii although orthology is not easy to define due to a high redundancy of the plasmid fraction. Yet, we did not find counterparts of the B.burgdorferi plasmids lp36 and lp38 or their respective gene repertoire in the B.garinii genome. Thus, phenotypic differences between the two species could be attributable to the presence or absence of these two plasmids as well as to the potentially positively selected genes.     

GuaA and GuaB Are Essential for Borrelia burgdorferi Survival in the Tick-Mouse Infection Cycle

Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.Purine metabolism is critical for the growth and virulence in mammals of many bacterial pathogens (11, 26, 29, 33, 51). Borrelia burgdorferi, the infectious agent of Lyme borreliosis, lacks the genes encoding the enzymes required for de novo nucleotide synthesis (8, 12) and therefore must rely on salvage of purines and pyrimidines from its hosts for nucleic acid biosynthesis (21, 35). Furthermore, B. burgdorferi lacks the genes encoding key enzymes required for a classic purine salvage pathway, including hpt (hypoxanthine-guanine phosphoribosyltransferase), purA (adenylosuccinate synthase), purB (adenylosuccinate lyase), and the locus encoding a ribonucleotide reductase (4, 8, 12, 35, 66). Despite the absence of a ribonucleotide reductase, an enzyme critical for the generation of deoxynucleotides through enzymatic reduction of ribonucleotides (32), a novel purine salvage pathway that involves salvage of deoxynucleosides from the host and interconversion of purine bases to deoxynucleosides by BB0426, a deoxyribosyl transferase, has recently been demonstrated for B. burgdorferi (23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pivotal role of the GuaAB proteins in the purine salvage pathway of B. burgdorferi. A novel pathway for purine salvage has recently been elucidated for B. burgdorferi (23). Extracellular adenine and hypoxanthine are salvaged by B. burgdorferi from mammalian and tick host environments (61). Following transport, adenine can be converted to hypoxanthine by adenine deaminase (BBK17) (21). This pathway proposes two possible fates for hypoxanthine, as follows. (i) Hypoxanthine is converted to IMP by a putative xanthine-guanine phosphoribosyl transferase (BB0103), IMP is converted to XMP by IMPDH (GuaB or BBB17), and XMP is converted to GMP by GMP synthase (GuaA or BBB18), resulting in guanine nucleotides for RNA synthesis. (ii) Direct transport of deoxynucleosides appears to provide a source of deoxyribose for interconversion of hypoxanthine to deoxyinosine by a deoxyribosyl transferase (BB0426) (23). dIMP is generated by a putative deoxynucleotide kinase (BB0239). GuaB converts dIMP to dXMP, and GuaA converts dXMP to dGMP, providing guanine deoxynucleotides for DNA synthesis (23). Salvage of free guanine nucleosides and guanine deoxynucleosides, when they are available in the host environment, may allow B. burgdorferi to circumvent the GuaAB requirement for GMP and dGMP biosynthesis. The dashed arrows indicate dephosphorylation of nucleotide monophosphate or deoxynucleotide monophosphate prior to transport by the spirochete and rephosphorylation of nucleoside and deoxynucleoside to nucleotide triphosphate and deoxynucleotide triphosphate, respectively, for RNA and DNA synthesis. NMP, nucleotide monophosphate; N, nucleoside; dN, deoxynucleoside; dNMP, deoxynucleotide monophosphate; OM, outer membrane; IM, inner membrane.In its infection cycle, B. burgdorferi passages between two disparate environments with potentially distinct purine availabilities, the tick vector and a mammalian host. Hypoxanthine is the most abundant purine in mammalian blood (17), and it is available for salvage by B. burgdorferi during the blood meal of an infected tick and during the spirochete''s transient presence in the mammalian bloodstream. Despite the absence of the hpt gene, we and others have shown that B. burgdorferi is able to transport and incorporate low levels of hypoxanthine (23, 35). During mammalian infection B. burgdorferi resides in various tissues, including the skin, heart, bladder, and joints. Adenine has been shown to be ubiquitous in mammalian tissues (61) and therefore is available for salvage by B. burgdorferi. Guanine is present at low levels in mammalian blood and tissues (17, 61); however, the amount may not be sufficient for survival of the spirochete.The limiting step in guanine nucleotide biosynthesis from adenine and hypoxanthine is the conversion of IMP to XMP, which is catalyzed by IMP dehydrogenase (IMPDH) (65). Guanine nucleotides are essential for DNA and RNA synthesis, signal transduction, and cell cycle control; thus, IMPDH activity is critical for the survival of most organisms (60). The enzymes required for the final two steps of guanine nucleotide biosynthesis, IMPDH and GMP synthase, are encoded by the guaB and guaA genes, respectively. The guaA and guaB genes and the corresponding activities of their protein products are conserved in B. burgdorferi (28, 67). These genes are typically carried on the chromosomes of bacterial species. However, in B. burgdorferi, the guaAB operon resides on a 26-kbp circular plasmid, cp26, and it shares an approximately 185-bp intergenic region with, and is transcribed divergently from, the essential virulence gene ospC (8, 12, 28, 50, 54, 62). The cp26 plasmid has been shown to harbor numerous genes important for B. burgdorferi survival in vivo and in vitro, including ospC (16, 34, 50, 53, 56) and resT (7), as well as BBB26 and BBB27 (20). Because of these critical functions, this plasmid is the only plasmid of the approximately 21 B. burgdorferi plasmids that is present in all natural isolates and has never been shown to be lost during in vitro culture (2, 7, 18, 20, 44, 52).Here we establish that the enzymatic activities of GuaA and GuaB are critical for the survival of B. burgdorferi in the infectious cycle and highlight a potential difference in this spirochete''s requirement for purine salvage in the disparate mammalian and tick environments.     

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.     

Intra- and Interbacterial Genetic Exchange of Lyme Disease Spirochete <Emphasis Type="Italic">erp</Emphasis> Genes Generates Sequence Identity Amidst Diversity

All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins. Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection. In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31. Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s. Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups. In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains. However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements. These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.     

Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch Migrase

Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et al. in the accompanying article, who also found a requirement for the RuvAB branch migrase. Our results also indicate that the mechanism of switching at vlsE in B. burgdorferi is distinct from switching at pilE in N. gonorrhoeae, which is the only other organism analyzed genetically in detail. Finally, our findings suggest a unique mechanism for switching at vlsE and a role for currently unidentified B. burgdorferi proteins in this process.     

Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi

Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.     

The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.     

Molecular Identification and Analysis of Borrelia burgdorferi Sensu Lato in Lizards in the Southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp− Escherichia coli

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.     

Transformation of the Lyme Disease Spirochete Borrelia burgdorferi with Heterologous DNA

Studies of the spirochete Borrelia burgdorferi have been hindered by the scarcity of genetic tools that can be used in these bacteria. For the first time, a method has been developed by which heterologous DNA (DNA without a naturally occurring B. burgdorferi homolog) can be introduced into and persistently maintained by B. burgdorferi. This technique uses integration of circular DNA into the bacterial genome via a single-crossover event. The ability to transform B. burgdorferi with heterologous DNA will now permit a wide range of experiments on the biology of these bacteria and their involvement in the many facets of Lyme disease.     

Pyruvate Protects Pathogenic Spirochetes from H2O2 Killing

Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H₂O₂ scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H₂O₂. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H₂O₂ (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H₂O₂ generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H₂O₂ challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H₂O₂ killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H₂O₂ killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H₂O₂ scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H₂O₂ generated by the host antibacterial response generated during infection.