Inflammation in the pathogenesis of lyme neuroborreliosis.

Lyme neuroborreliosis, caused by the spirochete Borrelia burgdorferi, affects both peripheral and central nervous systems. We assessed a causal role for inflammation in Lyme neuroborreliosis pathogenesis by evaluating the induced inflammatory changes in the central nervous system, spinal nerves, and dorsal root ganglia (DRG) of rhesus macaques that were inoculated intrathecally with live B. burgdorferi and either treated with dexamethasone or meloxicam (anti-inflammatory drugs) or left untreated. ELISA of cerebrospinal fluid showed significantly elevated levels of IL-6, IL-8, chemokine ligand 2, and CXCL13 and pleocytosis in all infected animals, except dexamethasone-treated animals. Cerebrospinal fluid and central nervous system tissues of infected animals were culture positive for B. burgdorferi regardless of treatment. B. burgdorferi antigen was detected in the DRG and dorsal roots by immunofluorescence staining and confocal microscopy. Histopathology revealed leptomeningitis, vasculitis, and focal inflammation in the central nervous system; necrotizing focal myelitis in the cervical spinal cord; radiculitis; neuritis and demyelination in the spinal roots; and inflammation with neurodegeneration in the DRG that was concomitant with significant neuronal and satellite glial cell apoptosis. These changes were absent in the dexamethasone-treated animals. Electromyography revealed persistent abnormalities in F-wave chronodispersion in nerve roots of a few infected animals; which were absent in dexamethasone-treated animals. These results suggest that inflammation has a causal role in the pathogenesis of acute Lyme neuroborreliosis.

Phosphoenolpyruvate Phosphotransferase System Components Modulate Gene Transcription and Virulence of Borrelia burgdorferi.

The phosphoenolpyruvate phosphotransferase system (PEP-PTS) and adenylate cyclase (AC) IV (encoded by BB0723 [cyaB]) are well conserved in different species of Borrelia. However, the functional roles of PEP-PTS and AC in the infectious cycle of Borrelia have not been characterized previously. We examined 12 PEP-PTS transporter component mutants by needle inoculation of mice to assess their ability to cause mouse infection. Transposon mutants with mutations in the EIIBC components (ptsG) (BB0645, thought to be involved in glucose-specific transport) were unable to cause infection in mice, while all other tested PEP-PTS mutants retained infectivity. Infectivity was partially restored in an in trans-complemented strain of the ptsG mutant. While the ptsG mutant survived normally in unfed as well as fed ticks, it was unable to cause infection in mice by tick transmission, suggesting that the function of ptsG is essential to establish infection by either needle inoculation or tick transmission. In Gram-negative organisms, the regulatory effects of the PEP-PTS are mediated by adenylate cyclase and cyclic AMP (cAMP) levels. A recombinant protein encoded by B. burgdorferi BB0723 (a putative cyaB homolog) was shown to have adenylate cyclase activity in vitro; however, mutants with mutations in this gene were fully infectious in the tick-mouse infection cycle, indicating that its function is not required in this process. By transcriptome analysis, we demonstrated that the ptsG gene may directly or indirectly modulate gene expression of Borrelia burgdorferi. Overall, the PEP-PTS glucose transporter PtsG appears to play important roles in the pathogenesis of B. burgdorferi that extend beyond its transport functions.

Antagonist of the neurokinin-1 receptor curbs neuroinflammation in ex vivo and in vitro models of Lyme neuroborreliosis.

BACKGROUND: Lyme neuroborreliosis (LNB) can affect both the peripheral (PNS) and the central nervous systems (CNS); it is caused by the spirochete Borrelia burgdorferi. The neuropeptide substance P (SP) is an important mediator of both neuroinflammation and blood-brain barrier dysfunction, through its NK1 receptor. Increased levels of SP have been shown to correlate with cell death. The present study used both ex vivo and in vitro models of experimentation to determine if the inflammatory mediator production and concomitant cell death caused by exposure of neural tissues and cells to B. burgdorferi could be attenuated by treatment with a NK1 receptor antagonist. METHODS: We incubated normal rhesus frontal cortex tissue explants (CNS) and primary cultures of rhesus dorsal root ganglia cells (PNS) with live B. burgdorferi and tested the effectiveness of the NK1 receptor antagonist L703,606 in attenuating inflammatory immune responses and neuronal and glial damage. Culture supernatants and tissue lysates were subjected to multiplex ELISA to quantify immune mediators, while the cells were evaluated for apoptosis by the in situ TUNEL assay. In addition, we identified immune mediators and producer cells in tissue sections by immunofluorescence staining and confocal microscopy. RESULTS: Co-incubation of both CNS tissues and PNS cells with the NK1 receptor antagonist attenuated bacterially induced increases in inflammatory cytokine and chemokine production, particularly, IL-6, CXCL8, and CCL2, and reduced apoptosis levels. Confocal microscopy confirmed that neurons and glial cells are sources of these immune mediators. These results suggest that NK1R antagonist treatment is able to reduce downstream pro-inflammatory signaling, thereby indicating that its systemic administration may slow disease progression. CONCLUSIONS: We propose that SP contributes to neurogenic inflammation in LNB, and provide data to suggest that an NK1 receptor antagonist may represent a novel neuroprotective therapy.

Superior efficacy of combination antibiotic therapy versus monotherapy in a mouse model of Lyme disease.

Lyme disease (LD) results from the most prevalent tick-borne infection in North America, with over 476,000 estimated cases annually. The disease is caused by Borrelia burgdorferi (Bb) sensu lato which transmits through the bite of Ixodid ticks. Most cases treated soon after infection are resolved by a short course of oral antibiotics. However, 10-20% of patients experience chronic symptoms because of delayed or incomplete treatment, a condition called Post-Treatment Lyme Disease (PTLD). Some Bb persists in PTLD patients after the initial course of antibiotics and an effective treatment to eradicate the persistent Bb is needed. Other organisms that cause persistent infections, such as M. tuberculosis, are cleared using a combination of therapies rather than monotherapy. A group of Food and Drug Administration (FDA)-approved drugs previously shown to be efficacious against Bb in vitro were used in monotherapy or in combination in mice infected with Bb. Different methods of detection were used to assess the efficacy of the treatments in the infected mice including culture, xenodiagnosis, and molecular techniques. None of the monotherapies eradicated persistent Bb. However, 4 dual combinations (doxycycline + ceftriaxone, dapsone + rifampicin, dapsone + clofazimine, doxycycline + cefotaxime) and 3 triple combinations (doxycycline + ceftriaxone+ carbomycin, doxycycline + cefotaxime+ loratadine, dapsone+ rifampicin+ clofazimine) eradicated persistent Bb infections. These results suggest that combination therapy should be investigated in preclinical studies for treating human Lyme disease.

Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva.

Uptake of the Lyme disease spirochete by its tick vector requires not only chemical signals present in the tick's saliva but a responsive phenotype by the Borrelia burgdorferi living in the mammalian host. This is the principle behind xenodiagnosis, wherein pathogen is detected by vector acquisition. To study migration of B. burgdorferi toward Ixodes scapularis tick saliva, with the goal of identifying chemoattractant molecules, we tested multiple assays and compared migration of host-adapted spirochetes to those cultured in vitro. We tested mammalian host-adapted spirochetes, along with those grown in culture at 34 °C, for their relative attraction to tick saliva or the nutrient N-acetyl-D-glucosamine (D-GlcNAc) and its dimer chitobiose using two different experimental designs. The host-adapted B. burgdorferi showed greater preference for tick saliva over the nutrients, whereas the cultured incubator-grown B. burgdorferi displayed no significant attraction to saliva versus a significant response to the nutrients. Our results not only describe a validated migration assay for studies of the Lyme disease agent, but provide a further understanding of how growth conditions and phenotype of B. burgdorferi are related to vector acquisition.

Central role of the Holliday junction helicase RuvAB in vlsE recombination and infectivity of Borrelia burgdorferi.

Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa surface-exposed lipoprotein, undergoes antigenic variation during B. burgdorferi infection of mammalian hosts, and is believed to be a critical mechanism by which the spirochetes evade immune clearance. Random, segmental recombination between the expressed vlsE gene and adjacent vls silent cassettes generates a large number of different VlsE variants within the infected host. Although the occurrence and importance of vlsE sequence variation is well established, little is known about the biological mechanism of vlsE recombination. To identify factors important in antigenic variation and vlsE recombination, we screened transposon mutants of genes known to be involved in DNA recombination and repair for their effects on infectivity and vlsE recombination. Several mutants, including those in BB0023 (ruvA), BB0022 (ruvB), BB0797 (mutS), and BB0098 (mutS-II), showed reduced infectivity in immunocompetent C3H/HeN mice. Mutants in ruvA and ruvB exhibited greatly reduced rates of vlsE recombination in C3H/HeN mice, as determined by restriction fragment polymorphism (RFLP) screening and DNA sequence analysis. In severe combined immunodeficiency (C3H/scid) mice, the ruvA mutant retained full infectivity; however, all recovered clones retained the 'parental' vlsE sequence, consistent with low rates of vlsE recombination. These results suggest that the reduced infectivity of ruvA and ruvB mutants is the result of ineffective vlsE recombination and underscores the important role that vlsE recombination plays in immune evasion. Based on functional studies in other organisms, the RuvAB complex of B. burgdorferi may promote branch migration of Holliday junctions during vlsE recombination. Our findings are consistent with those in the accompanying article by Dresser et al., and together these studies provide the first examples of trans-acting factors involved in vlsE recombination.

An immune evasion mechanism for spirochetal persistence in Lyme borreliosis.

Borrelia burgdorferi, the Lyme disease spirochete, persistently infects mammalian hosts despite the development of strong humoral responses directed against the pathogen. Here we describe a novel mechanism of immune evasion by B. burgdorferi. In immunocompetent mice, spirochetes that did not express ospC (the outer-surface protein C gene) were selected within 17 d after inoculation, concomitantly with the emergence of anti-OspC antibody. Spirochetes with no detectable OspC transcript that were isolated from immunocompetent mice reexpressed ospC after they were either cultured in vitro or transplanted to naive immunocompetent mice, but not in OspC-immunized mice. B. burgdorferi persistently expressed ospC in severe combined immune-deficient (SCID) mice. Passive immunization of B. burgdorferi-infected SCID mice with an anti-OspC monoclonal antibody selectively eliminated ospC-expressing spirochetes but did not clear the infection. OspC-expressing spirochetes reappeared in SCID mice after the anti-OspC antibody was eliminated. We submit that selection of surface-antigen nonexpressers is an immune evasion mechanism that contributes to spirochetal persistence.

Possible role of glial cells in the onset and progression of Lyme neuroborreliosis.

BACKGROUND: Lyme neuroborreliosis (LNB) may present as meningitis, cranial neuropathy, acute radiculoneuropathy or, rarely, as encephalomyelitis. We hypothesized that glia, upon exposure to Borrelia burgdorferi, the Lyme disease agent, produce inflammatory mediators that promote the acute cellular infiltration of early LNB. This inflammatory context could potentiate glial and neuronal apoptosis. METHODS: We inoculated live B. burgdorferi into the cisterna magna of rhesus macaques and examined the inflammatory changes induced in the central nervous system (CNS), and dorsal root nerves and ganglia (DRG). RESULTS: ELISA of the cerebrospinal fluid (CSF) showed elevated IL-6, IL-8, CCL2, and CXCL13 as early as one week post-inoculation, accompanied by primarily lymphocytic and monocytic pleocytosis. In contrast, onset of the acquired immune response, evidenced by anti-B. burgdorferi C6 serum antibodies, was first detectable after 3 weeks post-inoculation. CSF cell pellets and CNS tissues were culture-positive for B. burgdorferi. Histopathology revealed signs of acute LNB: severe multifocal leptomeningitis, radiculitis, and DRG inflammatory lesions. Immunofluorescence staining and confocal microscopy detected B. burgdorferi antigen in the CNS and DRG. IL-6 was observed in astrocytes and neurons in the spinal cord, and in neurons in the DRG of infected animals. CCL2 and CXCL13 were found in microglia as well as in endothelial cells, macrophages and T cells. Importantly, the DRG of infected animals showed significant satellite cell and neuronal apoptosis. CONCLUSION: Our results support the notion that innate responses of glia to B. burgdorferi initiate/mediate the inflammation seen in acute LNB, and show that neuronal apoptosis occurs in this context.

Multi-platform Approach for Microbial Biomarker Identification Using Borrelia burgdorferi as a Model.

The identification of microbial biomarkers is critical for the diagnosis of a disease early during infection. However, the identification of reliable biomarkers is often hampered by a low concentration of microbes or biomarkers within host fluids or tissues. We have outlined a multi-platform strategy to assess microbial biomarkers that can be consistently detected in host samples, using Borrelia burgdorferi, the causative agent of Lyme disease, as an example. Key aspects of the strategy include the selection of a macaque model of human disease, in vivo Microbial Antigen Discovery (InMAD), and proteomic methods that include microbial biomarker enrichment within samples to identify secreted proteins circulating during infection. Using the described strategy, we have identified 6 biomarkers from multiple samples. In addition, the temporal antibody response to select bacterial antigens was mapped. By integrating biomarkers identified from early infection with temporal patterns of expression, the described platform allows for the data driven selection of diagnostic targets.

Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete.

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

Variable manifestations, diverse seroreactivity and post-treatment persistence in non-human primates exposed to Borrelia burgdorferi by tick feeding.

The efficacy and accepted regimen of antibiotic treatment for Lyme disease has been a point of significant contention among physicians and patients. While experimental studies in animals have offered evidence of post-treatment persistence of Borrelia burgdorferi, variations in methodology, detection methods and limitations of the models have led to some uncertainty with respect to translation of these results to human infection. With all stages of clinical Lyme disease having previously been described in nonhuman primates, this animal model was selected in order to most closely mimic human infection and response to treatment. Rhesus macaques were inoculated with B. burgdorferi by tick bite and a portion were treated with recommended doses of doxycycline for 28 days at four months post-inoculation. Signs of infection, clinical pathology, and antibody responses to a set of five antigens were monitored throughout the ~1.2 year study. Persistence of B. burgdorferi was evaluated using xenodiagnosis, bioassays in mice, multiple methods of molecular detection, immunostaining with polyclonal and monoclonal antibodies and an in vivo culture system. Our results demonstrate host-dependent signs of infection and variation in antibody responses. In addition, we observed evidence of persistent, intact, metabolically-active B. burgdorferi after antibiotic treatment of disseminated infection and showed that persistence may not be reflected by maintenance of specific antibody production by the host.

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis.

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on naïve mice, all of which became infected as shown by culture of ear biopsy specimens. Naïve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.

Feeding of ticks on animals for transmission and xenodiagnosis in Lyme disease research.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Correction: Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection.

[This corrects the article on p. e29914 in vol. 7.].

Dynamic longitudinal antibody responses during Borrelia burgdorferi infection and antibiotic treatment of rhesus macaques.

Infection with Borrelia burgdorferi elicits robust yet disparate antibody responses in infected individuals. A longitudinal assessment of antibody responses to multiple diagnostic antigens following experimental infection and treatment has not previously been reported. Our goal was to identify a combination of antigens that could indicate infection at all phases of disease and response to antibiotic treatment. Because the rhesus macaque recapitulates the hallmark signs and disease course of human Lyme disease, we examined the specific antibody responses to multiple antigens of B. burgdorferi following infection of macaques. Five macaques infected with strain B31 and 12 macaques infected with strain JD1 were included in the analysis. Approximately half of these animals were treated with antibiotics at 4 to 6 months postinoculation. Antibody responses to several B. burgdorferi recombinant antigens, including OspC, DbpA, BBK32, OspA, and OppA-2, were measured at multiple points throughout infection. We have previously shown a decline in the response to the C6 peptide following antibiotic treatment. Responses to OspA and OspC, however, were variable over time among individuals, irrespective of antibiotic treatment. Not every individual responded to BBK32, but anti-DbpA IgG levels were uniformly high and remained elevated for all animals. All responded to OppA-2, with a decline posttreatment that was slow and incomplete. This is the first demonstration of B. burgdorferi OppA-2 antigenicity in nonhuman primates. The combination of DbpA, OspC, OspA, and OppA-2 with the C6 diagnostic peptide has the potential to detect infection throughout all disease phases.

Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity.

The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM) analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.

Dominant epitopes of the C6 diagnostic peptide of Borrelia burgdorferi are largely inaccessible to antibody on the parent VlsE molecule.

Lyme borreliosis (LB) is a disease for which antibody-based detection assays are often required for diagnosis. The variable surface molecule VlsE and IR6, one of its invariable regions, are commonly targeted by the antibody response in infected individuals. A series of enzyme-linked immunosorbent assays was performed to comparatively examine the antibody responses of North American LB patients (n = 37) to VlsE and invariable segments of this molecule. Both immunoglobulin M (IgM) and IgG responses to full-length VlsE and to peptides reproducing invariable regions 2, 4, and 6, as well as the invariable domains at the amino and carboxyl termini of VlsE, were assessed. The proportions and specificities of reactivity to the invariable segments were tested by using cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas <40% of patients generated antibodies that bound the N- or C-terminal domain and <12% of patients responded to either IR2 or IR4. Interestingly, 15 of 37 patients generated IgG antibodies that reacted with C6 but not with VlsE. Conversely, IgM responses were frequent for VlsE but not for invariable segments. A representative number of the serum specimens (n = 8) that contained IgG antibodies reacting with both C6 and VlsE was assessed in competition experiments, using C6 as a competitor. Only half of these specimens contained IgG antibodies whose binding to VlsE could be inhibited >50% by competition with the added C6 peptide. The median percent inhibition was 45.5%. These findings indicate that IR6 epitopes are largely concealed from the VlsE molecular surface and that full-length VlsE-based diagnosis likely detects antibodies to conformational and/or variable region epitopes.

Infectivity of the highly transformable BBE02- lp56- mutant of Borrelia burgdorferi, the Lyme disease spirochete, via ticks.

Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.