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

活性氧和氮对伯氏疏螺旋体基因调控的影响

• 批准号: 8156936
• 负责人: Frank Gherardini
• 金额: $ 51.97万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156936
• 关键词: Affect; Bacteria sigma factor KatF protein; Borrelia burgdorferi; DNA; DNA Damage; Data; Electrons; Environment; Enzymes; Escherichia coli; Eukaryotic Cell; Fatty Acids; Free Radicals; Gene Expression; Gene Expression Regulation; Homologous Gene; Nitrogen; O-(biotinylcarbazoylmethyl)hydroxylamine; Oxygen; Peroxonitrite; Process; Proliferating; Reactive Oxygen Species; Transcription Coactivator; Virulence Factors; biological adaptation to stress; defense response; in vivo; transmission process

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.

伯氏疏螺旋体是莱姆病的病原体，在节肢动物载体和各种哺乳动物宿主中存活并增殖。在其传播/感染周期中，伯氏疏螺旋体遇到了这些宿主特有的环境挑战。其中一项挑战来自活性氧 (ROS)，例如超氧自由基 (O2.-)、过氧化氢 (H2O2) 和羟基自由基 (OH.) 以及活性氮 (RNS)，例如一氧化氮 (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。第一个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使用多种机制（包括产生 ROS 和 RNS）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 Tom Schwan 博士合作，证明肩胛硬蜱的唾液腺含有显着水平的 ROS。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防御酶和促进生存和成功的关键毒力因子的表达。新宿主的殖民化。细胞对 ROS 破坏作用的防御涉及酶促和非酶促成分。 伯氏疏螺旋体具有有限数量的可能参与这种防御反应的酶。 已鉴定的酶包括 Mn 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 二硫化物还原酶 (CoADR)。 迄今为止，Mn-SOD、CoADR 和 NapA（未发表的数据）已通过实验进行了表征。 因为当受到来自宿主细胞的 O2.- 和 H2O2 的挑战时，这些酶会促进伯氏疏螺旋体细胞的体内存活，因此我们对该过程及其调节方式特别感兴趣。 疏螺旋体氧化应激调节因子 BosR 充当氧化应激基因的转录激活剂。 尽管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，我们还发现 BosR 调节 sodA (Mn-SOD)、编码 NapA（AhpR 同源物）的基因和 cdr (CoADR)。 ROS/RNS 对细胞的影响已被广泛研究。 这些高活性化合物已被证明会损害细胞大分子，包括 DNA、蛋白质和膜脂。在真核生物中，膜脂是活性氧的主要目标。 自由基攻击膜中的多不饱和脂肪酸并引发脂质过氧化。 主要影响是膜流动性降低，这会影响膜的物理特性，从而改变膜相关蛋白的功能。一旦脂质过氧化物形成，它们就会与邻近的多不饱和脂质发生反应，导致损害放大。 脂质过氧化物进一步氧化成多种产物，包括醛，随后与膜蛋白反应并损伤膜蛋白。然而，在细菌中，假设脂质不会受到真核细胞中观察到的氧化损伤。 只有某些多不饱和脂质，例如亚油酸和亚麻酸，才容易氧化，并且很明显，大多数细菌不会合成这些类型的脂质或将这些类型的脂质掺入其细胞膜中。 两个值得注意的例外是光合细菌和疏螺旋体属物种，它们在其膜中合成或掺入大量亚油酸和亚麻酸。 相反，事实证明，细菌中 ROS 最具破坏性的影响是由于自由基 (H2O2) 与“游离”Fe2+ 的相互作用，产生非常活泼的 OH-（芬顿反应）。由于这种反应性，它对任何给定的生物分子都会产生影响很大程度上取决于与目标的接近程度。 由于 Fe2+ 沿着核酸的磷酸二酯主链定位，因此 DNA 是 OH- 的主要目标。这种活性物质可以从碱基和糖部分拉出电子，产生各种损伤，包括主链中的单链和双链断裂以及化学交联到其他分子。 这些链断裂和其他阻碍 DNA 复制的损伤会导致 OH.- 毒性和细胞死亡。其他不妨碍复制的碱基损伤会导致突变率显着增加。伯氏疏螺旋体的细胞内生物化学表明，ROS 的主要细胞内靶标可能不是其他细菌（如大肠杆菌）中所描述的 DNA。在大肠杆菌中，H2O2 和芬顿化学造成的 DNA 损伤程度与细胞内的 Fe 代谢和游离 Fe 浓度成正比。 (5-100 nM) 由于伯氏疏螺旋体的细胞内 Fe 浓度估计为每个细胞 <10 个原子，DNA 似乎不太可能是伯氏疏螺旋体中 ROS 的主要目标。 为了支持这一点，伯氏疏螺旋体在 5mM H2O2 存在下的生长对 DNA 突变率几乎没有影响（自发性香豆霉素 A1 抗性）。 此外，当用各种氧化剂（叔丁基过氧化物或H2O2）处理细胞时，与未处理的细胞相比，没有检测到DNA损伤的增加，这是通过使用醛反应探针计算DNA碱基损伤的数量来确定的。 如前所述，伯氏疏螺旋体将环境中的多不饱和脂肪酸掺入细胞膜脂质和脂蛋白中，表明疏螺旋体膜可能是脂质过氧化的目标。 通过电子显微镜对叔丁基过氧化物处理的伯氏疏螺旋体细胞进行的分析显示出明显的不规则性，表明膜损伤。 对用叔丁基过氧化物和脂氧化酶处理的细胞进行的脂肪酸分析表明，这些细胞中源自宿主的亚油酸显着减少（分别是 10 倍和 50 倍），随后丙二醛 (MDA) 水平增加和 4-羟基烯醛 (HAE) 醛（分别是 4 倍和 10 倍），脂质过氧化的有毒副产物。 这些数据综合起来表明，伯氏疏螺旋体膜脂质和脂蛋白是感染周期各个阶段遇到的 ROS 攻击的主要目标，NapA 和 CoADR 可以清除细胞中的氧化脂质，并且编码这些脂质的基因蛋白质受 BosR 调节。