A Murine Model of Lyme Disease Demonstrates That Borrelia burgdorferi Colonizes the Dura Mater and Induces Inflammation in the Central Nervous System

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice are effective models for characterizing B. burgdorferi infection kinetics and host immune responses in joints and heart tissues; however, similar studies are lacking in the CNS of these animals. Here we characterize the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of infected C3H mice during early and subacute infection. B. burgdorferi colonized the dura mater following needle or tick challenge, and induced expression of inflammatory cytokines and a robust IFN response as well as histopathological changes. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines was unique to the brain parenchyma, and could provide insights into the mechanism of inflammatory CNS pathology associated with this important pathogen

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria

\u3cem\u3eBorrelia burgdorferi\u3c/em\u3e SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete

The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations

Altered murine tissue colonization by Borrelia burgdorferi following targeted deletion of linear plasmid 17-carried genes

The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined both in vitro and in vivo. A mutant strain lacking the genes bbd16 to bbd25 showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein C in vitro, which was correlated with the absence of the gene bbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone of B. burgdorferi and reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete

A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system.

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice have been used to define the kinetics of B. burgdorferi infection and host immune responses in joints and heart, however similar studies are lacking in the CNS of these animals. A tractable animal model for investigating host-Borrelia interactions in the CNS is key to understanding the mechanisms of CNS pathogenesis. Therefore, we characterized the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of mice during early and subacute infection. Using fluorescence-immunohistochemistry, intravital microscopy, bacterial culture, and quantitative PCR, we found B. burgdorferi routinely colonized the dura mater of C3H mice, with peak spirochete burden at day 7 post-infection. Dura mater colonization was observed for several Lyme disease agents including B. burgdorferi, B. garinii, and B. mayonii. RNA-sequencing and quantitative RT-PCR showed that B. burgdorferi infection was associated with increased expression of inflammatory cytokines and a robust interferon (IFN) response in the dura mater. Histopathologic changes including leukocytic infiltrates and vascular changes were also observed in the meninges of infected animals. In contrast to the meninges, we did not detect B. burgdorferi, infiltrating leukocytes, or large-scale changes in cytokine profiles in the cerebral cortex or hippocampus during infection; however, both brain regions demonstrated similar changes in expression of IFN-stimulated genes as observed in peripheral tissues and meninges. Taken together, B. burgdorferi is capable of colonizing the meninges in laboratory mice, and induces localized inflammation similar to peripheral tissues. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines is unique to the brain parenchyma, and provides insight into the potential mechanisms of CNS pathology associated with this important pathogen

Evaluation of the Importance of VlsE Antigenic Variation for the Enzootic Cycle of Borrelia burgdorferi

Efficient acquisition and transmission of Borrelia burgdorferi by the tick vector, and the ability to persistently infect both vector and host, are important elements for the life cycle of the Lyme disease pathogen. Previous work has provided strong evidence implicating the significance of the vls locus for B. burgdorferi persistence. However, studies involving vls mutant clones have thus far only utilized in vitro-grown or host-adapted spirochetes and laboratory strains of mice. Additionally, the effects of vls mutation on tick acquisition and transmission has not yet been tested. Thus, the importance of VlsE antigenic variation for persistent infection of the natural reservoir host, and for the B. burgdorferi enzootic life cycle in general, has not been examined to date. In the current work, Ixodes scapularis and Peromyscus maniculatus were infected with different vls mutant clones to study the importance of the vls locus for the enzootic cycle of the Lyme disease pathogen. The findings highlight the significance of the vls system for long-term infection of the natural reservoir host, and show that VlsE antigenic variability is advantageous for efficient tick acquisition of B. burgdorferi from the mammalian reservoir. The data also indicate that the adaptation state of infecting spirochetes influences B. burgdorferi avoidance from host antibodies, which may be in part due to its respective VlsE expression levels. Overall, the current findings provide the most direct evidence on the importance of VlsE for the enzootic cycle of Lyme disease spirochetes, and underscore the significance of VlsE antigenic variation for maintaining B. burgdorferi in nature

Differential Telomere Processing by Borrelia Telomere Resolvases In Vitro but Not In Vivo

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins

Total spirochete loads of <i>vls</i> mutant <i>B</i>. <i>burgdorferi</i>-infected <i>Ixodes scapularis</i>.

<p>Spirochetes in unfed nymphs were quantified by qPCR using a primer and internal probe for <i>flaB</i>. The number of spirochetes (log10) for each individual unfed tick is shown as an open circle. The black horizontal bar given for each <i>B</i>. <i>burgdorferi</i> group represents the overall mean. Only PCR-positive samples are included in this Fig. No statistical difference was observed between wtB31 (n = 15) and Δ<i>vlsE</i> (n = 12) or wtB31 (n = 15) and s<i>vlsE</i> (n = 13) in unfed nymphs, as determined by a two tailed t-test.</p

<i>vls</i> mutant clones do not show reduced spirochetemia in C3H mice.

<p>Blood was collected from saphenous and submandibular veins of C3H mice at day 7 post-infection after subcutaneous needle inoculation with <i>B</i>. <i>burgdorferi</i> clones. Isolated blood was plated in semi-solid BSK by limiting dilution, and colony-forming units (CFU) were enumerated for each strain. CFU/ml blood is shown for each mouse infected with wtB31 (closed circles), Δ<i>vlsE</i> (open circles), and s<i>vlsE</i> (closed triangles), as well as mean CFU/ml for each group (horizontal lines).</p

Design of passive transfer experiment to assess the ability of <i>B</i>. <i>burgdorferi</i> clones to evade antibodies.

<p>Preimmune and immune sera were originated, respectively, from naïve and Δ<i>vlsE</i>-infected <i>P</i>. <i>maniculatus</i> mice. Four groups of SCID mice (3 animals per group) were treated with immune sera from Δ<i>vlsE</i>-infected mice and challenged 18 hours later with host-adapted wtB31 (group I), host-adapted Δ<i>vlsE</i> (group II), nymph-transmitted wtB31 (group III) or nymph-transmitted Δ<i>vlsE</i> (group IV) clones. At day 7 post infection, blood was drawn from each mouse and cultured to assess the outcome of challenge. The control groups of mice confirmed the ability of host-adapted Δ<i>vlsE</i> or nymph-transmitted wtB31 and Δ<i>vlsE</i> clones to infect preimmune sera-treated or naïve SCID mice, respectively.</p