Telomere resolution in the Lyme disease spirochete.
(9/1041)
The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase. (+info)
Demonstration of the genetic stability and temporal expression of select members of the lyme disease spirochete OspF protein family during infection in mice.
(10/1041)
Infection with Lyme disease spirochetes can be chronic. This suggests that the spirochetes are capable of immune evasion. In a previous study we demonstrated that the ospE gene family, which is one of three gene families whose members are flanked at their 5' end by the highly conserved upstream homology box (UHB) element, undergoes mutation and rearrangement during infection. This results in the generation of antigenically distinct variants that may contribute to immune evasion. In this study we have assessed the genetic stability of the UHB-flanked ospF gene family during infection in mice. Using postinfection clonal populations of Borrelia burgdorferi B31MI, PCR amplicons were generated for three members of the ospF gene family after a 3-month infection time frame. The amplicons were analyzed by single-nucleotide polymorphism pattern analysis and DNA sequencing. Members of the ospF gene family were found to be stable during infection, as no mutations or rearrangements were detected. An analysis of the humoral immune response to these proteins during infection revealed that the immune response to each is specific and that there is a delayed humoral immune response to some OspF protein family members. These analyses suggest that there is a temporal component to the expression of these genes during infection. In addition to a possible contribution to immune evasion, members of the OspF protein family may play specific roles at different stages of infection. (+info)
Use of serum immune complexes in a new test that accurately confirms early Lyme disease and active infection with Borrelia burgdorferi.
(11/1041)
The present recommendation for serologic confirmation of Lyme disease (LD) calls for immunoblotting in support of positive or equivocal ELISA. Borrelia burgdorferi releases large quantities of proteins, suggesting that specific antibodies in serum might be trapped in immune complexes (ICs), rendering the antibodies undetectable by standard assays using unmodified serum. Production of ICs requires ongoing antigen production, so persistence of IC might be a marker of ongoing or persisting infection. We developed an immunoglobulin M (IgM) capture assay (EMIBA) measuring IC-derived IgM antibodies and tested it using three well-defined LD populations (from an academic LD referral center, a well-described Centers for Disease Control and Prevention (CDC) serum bank, and a group of erythema migrans patients from whose skin lesions B. burgdorferi was grown) and controls (non-Lyme arthritis inflammatory joint disease, syphilis, multiple sclerosis, and nondisease subjects from a region where LD is endemic, perhaps the most relevant comparison group of all). Previous studies demonstrated that specific antigen-antibody complexes in the sera of patients with LD could be precipitated by polyethylene glycol and could then be disrupted with maintenance of the immunoreactivity of the released antibodies, that specific anti-B. burgdorferi IgM was concentrated in ICs, and that occasionally IgM to specific B. burgdorferi antigens was found in the IC but not in unprocessed serum. EMIBA compared favorably with commercial and CDC flagellin-enhanced enzyme-linked immunosorbent assays and other assays in confirming the diagnosis of LD. EMIBA confirmed early B. burgdorferi infection more accurately than the comparator assays. In addition, EMIBA more accurately differentiated seropositivity in patients with active ongoing infection from seroreactivity persisting long after clinically successful antibiotic therapy; i.e., EMIBA identified seroreactivity indicating a clinical circumstance requiring antibiotic therapy. Thus, EMIBA is a promising new assay for accurate serologic confirmation of early and/or active LD. (+info)
Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination.
(12/1041)
Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-gammaR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-gamma-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-gamma and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-gamma-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-gamma-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-gammaR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi. (+info)
Yield of large-volume blood cultures in patients with early Lyme disease.
(13/1041)
To improve yield, 6 3-mL plasma cultures (18 mL total) were established for adult patients with early Lyme disease associated with erythema migrans. Borrelia burgdorferi was recovered from the blood of 22 (44.0%) of 50 evaluable patients. The recovery rate per plasma culture and the frequency of positive results for plasma cultures for individual patients were consistent with a level of spirochetemia of approximately 0.1 cultivable cell/mL of whole blood. Our findings suggest that, if further improvements in the yield of blood cultures are possible, they probably will depend on enhancing the sensitivity of the culture method rather than increasing the volume of material cultured. (+info)
Analysis of Borrelia burgdorferi vlsE gene expression and recombination in the tick vector.
(14/1041)
Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34 degrees C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector. (+info)
Likelihoods and simulation methods for a class of nonneutral population genetics models.
(15/1041)
Methods for simulating samples and sample statistics, under mutation-selection-drift equilibrium for a class of nonneutral population genetics models, and for evaluating the likelihood surface, in selection and mutation parameters, are developed and applied for observed data. The methods apply to large populations in settings in which selection is weak, in the sense that selection intensities, like mutation rates, are of the order of the inverse of the population size. General diploid selection is allowed, but the approach is currently restricted to models, such as the infinite alleles model and certain K-models, in which the type of a mutant allele does not depend on the type of its progenitor allele. The simulation methods have considerable advantages over available alternatives. No other methods currently seem practicable for approximating likelihood surfaces. (+info)
Complementation of a nonmotile flaB mutant of Borrelia burgdorferi by chromosomal integration of a plasmid containing a wild-type flaB allele.
(16/1041)
With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed. (+info)