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. Lyme disease, caused by Borrelia burgdorferi, is a complex vector-borne infection that causes a multistage inflammatory disease in numerous systems resulting in significant morbidity 1-4. The multiple stages begin with a localized dermal colonization followed by a dissemination to distal immunoprotective niches, including the central nervous system (CNS), heart, and joints, that can result in the development of neuroborreliosis, Lyme carditis, and/or arthritis, respectively. A robust innate and adaptive immune response is elicited by B. burgdorferi that is unable to clear infection or prevent the progression of disease 1. Antibiotic treatment effectively resolves the disease during early localized infection, but less so after dissemination and late Lyme disease. The Center for Disease Control (CDC) recently reported a substantial increase in the number of cases in the United States from approximately 30,000 to 300,000 per year due to under reporting and less than optimal diagnostic tools 5. Lyme disease is a significant public health concern with limited treatment options to protect and maintain long term quality of life in humans. Borrelia burgdorferi, a spirochetal bacterium, dynamically regulates gene expression to promote each stage of disease in the vector and mammalian host modulate disease and evade the immune response 2,6-8. Changes in environmental cues, such as pH, temperature, O 2 , CO 2 , metals and osmotic stress, detected by B. burgdorferi result in differential presentation of lipoproteins on the outer membrane to promote adherence, invasion, and colonization 9-17. Unknown host specific and tissue specific signals in...

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3′ end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full length SR0623 to evaluate the influence on transcriptional and post-transcriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.