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Lyme disease, caused by Borrelia burgdorferi, is an inflammatory multistage infection, consisting of localized, disseminated, and persistent disease stages, impacting several organ systems through poorly defined gene regulation mechanisms. The purpose of this study is to further characterize the spatiotemporal transcriptional regulation of B. burgdorferi during mammalian infection of borrelial oxidative stress regulator (bosR) and decorin binding protein (dbpBA) by utilizing bioluminescent B. burgdorferi reporter strains and in vivo imaging. Fluctuating borrelial load was also monitored and used for normalization to evaluate expression levels. bosR transcription is driven by two promoters, P bb0648 and P bosR , and we focused on the native promoter. bosR expression is low relative to the robustly expressed dbpBA throughout infection. In distal tissues, bosR was the highest in the heart during in the first week whereas dbpBA was readily detectable at all time points with each tissue displaying a distinct expression pattern. This data suggests bosR may have a role in heart colonization and the induction of dbpBA indicates a RpoS independent transcriptional regulation occurring in the mammalian cycle of pathogenesis. These finding demonstrate that B. burgdorferi engages unknown genetic mechanisms to uniquely respond to mammalian tissue environments and/or changing host response over time.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction., Competing Interests: The authors have declared that no competing interests exist.

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2-RpoN-RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS-dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2-RpoN-RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33-dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance., (© 2015 John Wiley & Sons Ltd.)

Summary Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low-passage B. burgdorferi isolate was constructed. The resulting bosR::kan(R) strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2-RpoN-RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non-infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.

Borrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kan(R)) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kan(R) strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macroarray technology and quantitative RT-PCR were utilized to compare the transcriptional profiles from the isogenic bosRR39K and bosRR39K : : kan(R) isolates. Array data indicated that 88 ORFs were significantly expressed in the absence of BosRR39K. Since most affected genes mapped to the chromosome, it is likely that these genes define an important physiologic response for B. burgdorferi. Included within the genes identified was the detoxification gene sodA, as well as other loci not overtly linked to oxidative stress. These results suggest that a putative BosR regulon, as defined by the bosRR39K allele, is required to combat toxic oxidative intermediates, but may also be involved in adaptive strategies that are independent of reactive oxygen species.

Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.