Summary The etiologic agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post-infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify, and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.

SummaryBorrelia 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.

The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO 2 , serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO 2 , 3% H 2 , 0.087 ppm O 2 ) and microaerophilic (1% CO 2 , 3.48 ppm O 2 ) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO 2 . In this study we identified CO 2 as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO 2 , independent of O 2 levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO 2 . Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO 2 , indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO 2 may be an additional cue for borrelial host adaptation and gene regulation.

SummaryBorrelia 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.