Borrelia burgdorferi, the causative agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One such challenge comes from reactive oxygen species (ROS) [e.g. superoxide radicals (O2.-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH.?)] and reactive nitrogen species (RNS) [e.g. nitric oxide (NO) and peroxynitrite]. There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs as the bacteria migrate through the salivary glands during transmission. Our lab, in collaboration with Dr. Tom Schwan, has demonstrated that the salivary glands of Ixodes scapularis contain significant levels of ROS. Therefore, our current working model is that as B. burgdorferi migrates from the anaerobic midgut (containing no ROS) to the salivary glands, ROS act as a signal to induce the expression of ROS defense enzymes and key virulence factors that promote the survival and successful colonization of a new host. Cellular defenses against the damaging effects of ROS involve both enzymatic and nonenzymatic components. B. burgdorferi has a limited number of enzymes that could potentially be involved in this defense response. Those identified include a Mn-dependent superoxide dismutase (SOD), a Dps/Dpr homologue (NapA), thioredoxin (Trx), thioredoxin reductase (TrxR) and a Coenzyme-A disulfide reductase (CoADR). To date the Mn-SOD, CoADR, and NapA (unpublished data) have been characterized experimentally. Because these enzymes would promote the in vivo survival of B. burgdorferi cells when challenged by O2.- and H2O2 from host cells, we are particularly interested in the process and how it is regulated. The Borrelia oxidative stress regulator, BosR, acts as a transcriptional activator of oxidative stress genes. Although there is no apparent amino acid homology (>15%), BosR appears to be functionally similar to OxyR. In addition, we have shown that BosR regulates sodA (Mn-SOD), the gene encoding NapA (an AhpR homolog), and cdr (CoADR). Our working hypothesis is that B. burgdorferi cells are using these proteins to rid the cells of damaging oxidants and maintain intracellular redox in a process that is regulated by BosR.? ? OBJECTIVES: To continue to characterize the oxidative stress response in B. burgdorferi we will: (i) continue the biochemical characterization of NapA and CoADR, and identify additional genes in the BosR regulon using microarrays; (ii) determine stage of the infective cycle when BosR regulated genes are expressed using GFP fusions to track the expression of genes from midgut, through the salivary glands, and into the mammalian host; and (iii) because we have been unable to generate mutants in bosR, napA or cdr, we will develop additional genetic tools (e.g., a RNA silencing system on pBSV2 with an inducible promoter) to study genes encoding proteins with essential functions in B. burgdorferi.
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