The Effects of Reactive Oxygen and Nitrogen on Gene Regulation in B. Burgdorferi

伯氏疏螺旋体，莱姆病的病原体，在节肢动物媒介和各种哺乳动物宿主中存活和增殖。在其传播/感染周期中，伯氏疏螺旋体会遇到这些宿主特有的环境挑战。其中一个挑战来自活性氧（ROS），如超氧自由基（O2）。-)、过氧化氢（H2O2）和羟基自由基（OH）以及活性氮物质（RNS），如一氧化氮（NO）、N2O3和过氧亚硝酸盐。当伯氏疏螺旋体暴露于ROS/RNS时，感染周期分为两个阶段。第一种是在哺乳动物宿主感染的初始阶段，此时免疫系统细胞试图通过几种机制，包括ROS和RNS的产生，来限制和消除伯氏疏螺旋体。令人惊讶的是，第二次ROS/RNS挑战发生在细菌在传播过程中通过唾液腺迁移时。我们的实验室，与汤姆·施万博士合作，已经证明肩胛棘猴的唾液腺含有大量的活性氧。因此，我们目前的工作模型是，当伯氏疏螺旋体从厌氧中肠（不含ROS）迁移到唾液腺时，ROS作为信号诱导ROS防御酶和关键毒力因子的表达，促进新宿主的生存和成功定殖。细胞防御ROS的破坏作用包括酶和非酶成分。伯氏疏螺旋体有有限数量的酶可能参与这种防御反应。这些鉴定包括锰依赖性超氧化物歧化酶（SOD）， Dps/Dpr同源物（NapA），硫氧还蛋白（Trx），硫氧还蛋白还原酶（TrxR）和辅酶- a二硫还原酶（CoADR）。迄今为止，Mn-SOD、CoADR和NapA（未发表的数据）已被实验表征。因为这些酶能促进伯氏疏螺旋体细胞在缺氧条件下的存活。-和H2O2，我们对这一过程及其调控方式特别感兴趣。疏螺旋体氧化应激调节因子BosR是氧化应激基因的转录激活因子。虽然没有明显的氨基酸同源性（>15%），BosR似乎在功能上与大肠杆菌中的OxyR相似。此外，我们已经证明BosR调节sodA (Mn-SOD)，编码NapA （AhpR同源物）和cdr （CoADR）的基因。

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), N2O3 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 from E. coli. In addition, we have shown that BosR regulates sodA (Mn-SOD), the gene encoding NapA (an AhpR homolog), and cdr (CoADR). The effects of ROS/RNS on cells have been extensively investigated. These highly reactive compounds have been shown to damage cellular macromolecules including DNA, proteins, and membrane lipids. In eukaryotes, membrane lipids are a major target of reactive oxygen species. Free radicals attack polyunsaturated fatty acids in membranes and initiate lipid peroxidation. A primary effect is a decrease in membrane fluidity which affects the physical properties of the membrane altering the function of membrane-associated proteins. Once lipid peroxides form, they react with adjacent polyunsaturated lipids causing an amplification of the damage. Lipid peroxides undergo further oxidation to a variety of products, including aldehydes, which subsequently react with and damage membrane proteins. However, in bacteria, it is assumed that lipids are not subject to the oxidative damage observed in eukaryotic cells. Only certain polyunsaturated lipids, such as linoleic and linolenic acid, are susceptible to oxidation and it is clear that most bacteria do not synthesize or incorporate these types of lipids in their cell membranes. Two notable exceptions are the photosynthetic bacteria and Borrelia species who synthesize or incorporate significant levels of linoleic and linolenic acid in their membranes. Instead, it has been shown that the most damaging effects of ROS in bacteria result from the interactions of radicals (H2O2) with "free" Fe2+ generating very reactive OH- (Fenton reaction).Because of this reactivity, its effect on any given biomolecule will depend largely upon proximity to the target. Because Fe2+ localizes along the phosphodiester backbone of nucleic acid, DNA is a major target of OH-.This reactive species can pull electrons from either the base and sugar moieties producing a varety of lesions including single and double strand breaks in the backbone and chemical crosslinks to other molecules. These strand breaks, and other lesions that block DNA replication, contribute to OH.- toxicity and cell death. Other base damage, that does not hinder replication, contributes to a significant increase in mutation rates. The intracellular biochemistry of B. burgdorferi suggest that the primary intracellular target of ROS may not be DNA as described in other bacteria such as E. coli. In E.coli, the extent of DNA damage due to H2O2 and Fenton chemistry is directly proportional to Fe metabolism and the free Fe concentration within the cell.(5-100 nM) Since the intracellular Fe concentrations of B. burgdorferi are estimated to be <10 atoms per cell, it seems unlikely that DNA is a primary target for ROS in B. burgdorferi. In support of this, growth of B. burgdorferi in the presence of 5mM H2O2 had little to no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance). Also, when cells were treated with various oxidants (t-butyl peroxide or H2O2) no increase in DNA damage was detected in comparison to untreated cells as determined by calculating the number of DNA base lesions using an aldehyde reactive probe. As previously mentioned, B. burgdorferi incorporates polyunsaturated fatty acids from the environment into the cells membrane lipids and lipoproteins suggesting that Borrelia membranes could be a target for lipid peroxidation. Analyses of t-butyl peroxide treated B. burgdorferi cells by electron microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with t-butyl peroxide and lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (10- and 50-fold, respectively) in these cells, with a subsequent increase in the levels of malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) aldehyde(4- and 10-fold respectively),the toxic by-products of lipid peroxidation. These data, taken together, suggest that B. burgdorferi membrane lipids and lipoproteins are the primary targets for attack by ROS encountered in the various stages of the infective cycle, that NapA and CoADR rid the cells of oxidized lipids, and that the genes encoding these proteins are regulated by BosR.