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When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

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. IMPORTANCE B. burgdorferi persists in nature by cycling between ticks and vertebrates. Little is known about how the bacterium senses and adapts to each niche of the cycle. The present studies indicate that B. burgdorferi controls production of SpoVG and that this protein binds to specific sites of DNA and RNA in the genome and transcriptome, respectively. Altered expression of spoVG exerts effects on bacterial replication and other aspects of the spirochete's physiology., (Copyright © 2018 American Society for Microbiology.)

Unlabelled: In many bacteria, the FtsH protease and its modulators, HflK and HflC, form a large protein complex that contributes to both membrane protein quality control and regulation of the cellular response to environmental stress. Both activities are crucial to the Lyme disease pathogen Borrelia burgdorferi, which depends on membrane functions, such as motility, protein transport, and cell signaling, to respond to rapid changes in its environment. Using an inducible system, we demonstrate that FtsH production is essential for both mouse and tick infectivity and for in vitro growth of B. burgdorferi FtsH depletion in B. burgdorferi cells resulted in membrane deformation and cell death. Overproduction of the protease did not have any detectable adverse effects on B. burgdorferi growth in vitro, suggesting that excess FtsH does not proteolytically overwhelm its substrates. In contrast, we did not observe any phenotype for cells lacking the protease modulators HflK and HflC (ΔHflK/C), although we examined morphology, growth rate, growth under stress conditions, and the complete mouse-tick infectious cycle. Our results demonstrate that FtsH provides an essential function in the life cycle of the obligate pathogen B. burgdorferi but that HflK and HflC do not detectably affect FtsH function., Importance: Lyme disease is caused by Borrelia burgdorferi, which is maintained in nature in an infectious cycle alternating between small mammals and Ixodes ticks. B. burgdorferi produces specific membrane proteins to successfully infect and persist in these diverse organisms. We hypothesized that B. burgdorferi has a specific mechanism to ensure that membrane proteins are properly folded and biologically active when needed and removed if improperly folded or dysfunctional. Our experiments demonstrate that FtsH, a protease that fulfills this role in other microorganisms, is essential to B. burgdorferi viability. Cells depleted of FtsH do not survive in laboratory culture medium and cannot colonize mice or ticks, revealing an absolute requirement for this protease. However, the loss of two potential modulators of FtsH activity, HflK and HflC, does not detectably affect B. burgdorferi physiology. Our results provide the groundwork for the identification of FtsH substrates that are critical for the bacterium's viability., (Copyright © 2016 Chu et al.)

Borrelia burgdorferi is a zoonotic pathogen whose maintenance in nature depends upon an infectious cycle that alternates between a tick vector and mammalian hosts. Lyme disease in humans results from transmission of B. burgdorferi by the bite of an infected tick. The population dynamics of B. burgdorferi throughout its natural infectious cycle are not well understood. We addressed this topic by assessing the colonization, dissemination and persistence of B. burgdorferi within and between the disparate mammalian and tick environments. To follow bacterial populations during infection, we generated seven isogenic but distinguishable B. burgdorferi clones, each with a unique sequence tag. These tags resulted in no phenotypic changes relative to wild type organisms, yet permitted highly sensitive and specific detection of individual clones by PCR. We followed the composition of the spirochete population throughout an experimental infectious cycle that was initiated with a mixed inoculum of all clones. We observed heterogeneity in the spirochete population disseminating within mice at very early time points, but all clones displayed the ability to colonize most mouse tissues by 3 weeks of infection. The complexity of clones subsequently declined as murine infection persisted. Larval ticks typically acquired a reduced and variable number of clones relative to what was present in infected mice at the time of tick feeding, and maintained the same spirochete population through the molt to nymphs. However, only a random subset of infectious spirochetes was transmitted to naïve mice when these ticks next fed. Our results clearly demonstrate that the spirochete population experiences stochastic bottlenecks during both acquisition and transmission by the tick vector, as well as during persistent infection of its murine host. The experimental system that we have developed can be used to further explore the forces that shape the population of this vector-borne bacterial pathogen throughout its infectious cycle.

The Lyme disease spirochete, Borrelia burgdorferi, exists in a zoonotic cycle involving an arthropod tick and mammalian host. Dissemination of the organism within and between these hosts depends upon the spirochete's ability to traverse through complex tissues. Additionally, the spirochete outruns the host immune cells while migrating through the dermis, suggesting the importance of B. burgdorferi motility in evading host clearance. B. burgdorferi's periplasmic flagellar filaments are composed primarily of a major protein, FlaB, and minor protein, FlaA. By constructing a flaB mutant that is nonmotile, we investigated for the first time the absolute requirement for motility in the mouse-tick life cycle of B. burgdorferi. We found that whereas wild-type cells are motile and have a flat-wave morphology, mutant cells were nonmotile and rod shaped. These mutants were unable to establish infection in C3H/HeN mice via either needle injection or tick bite. In addition, these mutants had decreased viability in fed ticks. Our studies provide substantial evidence that the periplasmic flagella, and consequently motility, are critical not only for optimal survival in ticks but also for infection of the mammalian host by the arthropod tick vector.

Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector, Ixodes ticks, and a diverse range of vertebrate hosts. B. burgdorferi linear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for the B. burgdorferi infectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in the B. burgdorferi infectious cycle, we constructed a shuttle vector to selectively displace lp38 from the B. burgdorferi genome and analyzed the resulting clones to confirm the loss of lp38. We found that, in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.

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.

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.

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.

We previously demonstrated that Borrelia burgdorferi requires OspC to colonize a mammalian host. To delineate this requirement, we analyzed the clearance of ospC mutant spirochetes and found that they were eliminated within 48 h. We conclude that B. burgdorferi uses OspC to resist innate host defenses immediately after transmission.

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.

Background: Borrelia burgdorferi, the tick-transmitted agent of Lyme disease, adapts to different environments as it cycles between an arthropod vector and vertebrate host. Signals encountered during nymphal tick feeding prior to transmission activate a regulon that is controlled by the alternative sigma factors RpoN and RpoS, which are required for mammalian infection. The ingested bloodmeal also provides nutrients that stimulate spirochete replication. Although the influence of tick feeding on spirochete growth and gene expression is well documented, a quantitative assessment of spirochete virulence before and after tick feeding has not been made. Methods: Homogenates were prepared from unfed and fed infected Ixodes scapularis nymphs that had acquired B. burgdorferi as larvae. Serially diluted tick homogenates were needle-inoculated into mice to determine the infectious dose of tick-derived spirochetes before and after the bloodmeal. Mouse infection was assessed by sero-reactivity with B. burgdorferi whole cell lysates on immunoblots and attempted isolation of spirochetes from mouse tissues. Viable spirochetes in tick-derived inocula were quantified by colony formation in solid media. Results: We found that an inoculum containing as many as 104 B. burgdorferi from unfed ticks is largely non-infectious, while the calculated ID50 for spirochetes from fed ticks is ∼30 organisms. Engineered constitutive production of the essential virulence factor OspC by spirochetes within unfed ticks did not confer an infectious phenotype. Conclusion: Conditional priming of B. burgdorferi during tick feeding induces changes in addition to OspC that are required for infection of the mammalian host. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi is a zoonotic pathogen whose maintenance in nature depends upon an infectious cycle that alternates between a tick vector and mammalian hosts. Lyme disease in humans results from transmission of B. burgdorferi by the bite of an infected tick. The population dynamics of B. burgdorferi throughout its natural infectious cycle are not well understood. We addressed this topic by assessing the colonization, dissemination and persistence of B. burgdorferi within and between the disparate mammalian and tick environments. To follow bacterial populations during infection, we generated seven isogenic but distinguishable B. burgdorferi clones, each with a unique sequence tag. These tags resulted in no phenotypic changes relative to wild type organisms, yet permitted highly sensitive and specific detection of individual clones by PCR. We followed the composition of the spirochete population throughout an experimental infectious cycle that was initiated with a mixed inoculum of all clones. We observed heterogeneity in the spirochete population disseminating within mice at very early time points, but all clones displayed the ability to colonize most mouse tissues by 3 weeks of infection. The complexity of clones subsequently declined as murine infection persisted. Larval ticks typically acquired a reduced and variable number of clones relative to what was present in infected mice at the time of tick feeding, and maintained the same spirochete population through the molt to nymphs. However, only a random subset of infectious spirochetes was transmitted to naïve mice when these ticks next fed. Our results clearly demonstrate that the spirochete population experiences stochastic bottlenecks during both acquisition and transmission by the tick vector, as well as during persistent infection of its murine host. The experimental system that we have developed can be used to further explore the forces that shape the population of this vector-borne bacterial pathogen throughout its infectious cycle. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi is a zoonotic pathogen whose maintenance in nature depends upon an infectious cycle that alternates between a tick vector and mammalian hosts. Lyme disease in humans results from transmission of B. burgdorferi by the bite of an infected tick. The population dynamics of B. burgdorferi throughout its natural infectious cycle are not well understood. We addressed this topic by assessing the colonization, dissemination and persistence of B. burgdorferi within and between the disparate mammalian and tick environments. To follow bacterial populations during infection, we generated seven isogenic but distinguishable B. burgdorferi clones, each with a unique sequence tag. These tags resulted in no phenotypic changes relative to wild type organisms, yet permitted highly sensitive and specific detection of individual clones by PCR. We followed the composition of the spirochete population throughout an experimental infectious cycle that was initiated with a mixed inoculum of all clones. We observed heterogeneity in the spirochete population disseminating within mice at very early time points, but all clones displayed the ability to colonize most mouse tissues by 3 weeks of infection. The complexity of clones subsequently declined as murine infection persisted. Larval ticks typically acquired a reduced and variable number of clones relative to what was present in infected mice at the time of tick feeding, and maintained the same spirochete population through the molt to nymphs. However, only a random subset of infectious spirochetes was transmitted to naïve mice when these ticks next fed. Our results clearly demonstrate that the spirochete population experiences stochastic bottlenecks during both acquisition and transmission by the tick vector, as well as during persistent infection of its murine host. The experimental system that we have developed can be used to further explore the forces that shape the population of this vector-borne bacterial pathogen throughout its infectious cycle. [ABSTRACT FROM AUTHOR]

Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice. [ABSTRACT FROM AUTHOR]