Characterization Of The Oxidative Stress Response In Bor

由于致病菌产生的重要毒力因子（毒素、黏附素等）通常受到环境信号（温度、pH、离子/金属浓度等）的调节，我们已经开始在伯氏疏螺旋体中识别这些调节系统。SDS-PAGE和免疫印迹分析表明，在EDDHA、2-2巯基（DIP）或chelex等金属螯合剂处理过的修饰barbur - stoenner - kelly （BSK-II）培养基中，伯氏疏螺旋体生长的蛋白质会随着金属浓度的降低而改变蛋白质的表达。已确定的调节系统是基于金属依赖的抑制蛋白，它可以感知细胞内铁的浓度。最初，我们认为这是伯氏疏螺旋体的情况。然而，一些实验结果强烈表明，伯氏疏螺旋体对铁的需求很低或不存在。这些发现以及伯氏疏螺旋体缺乏呼吸链的事实表明，在正常细胞生长过程中，代谢不会产生活性氧（ROS）。同样重要的是，没有细胞内铁，就没有与ROS，特别是H2O2的芬顿反应。因此，对伯氏疏螺旋体细胞的氧化挑战必须主要以ROS和/或活性氮（NOS）的形式来自宿主。为了了解伯氏疏螺旋体如何生化消除或减少ROS（或NOS）并调节参与这一过程的酶，我们集中研究了三种蛋白质。我们从伯氏疏螺旋体中克隆了一个假定的金属依赖性抑制蛋白（PerR）的基因，并利用迁移位移dna结合试验鉴定了两个靶序列。其中一个序列位于已知的编码谷氨酸转运蛋白（gltP）和NADH过氧化物酶（npx）的2基因操纵子的起始密码子上游60 bp处。另一个位于napA起始密码子上游57bp处，napA基因编码烷基过氧化氢还原酶。这些数据表明PerR可能参与调节伯氏疏螺旋体的氧化应激反应。从枯草芽孢杆菌中鉴定出的PerR同源物介导该细菌对氧化应激和金属饥饿的细胞反应。显然，表征该调节蛋白在伯氏疏螺旋体生存反应中的作用并确定其调节的其他基因将有助于对莱姆病的理解。到目前为止，我们已经完成了以下工作：(1)为了开始对该系统进行表征，我们将伯氏螺旋体中的perR克隆到表达载体pKK223-3中，用IPTG诱导，并从大肠杆菌XL1-Blue MRF&#697中纯化该蛋白。用肝素-琼脂糖亲和柱纯化PerR，制备重组蛋白的多克隆血清。该抗血清在伯氏疏螺旋体细胞裂解物中检测到PerR，并用于检测高传代PerR突变体。(2)从伯氏疏螺旋体基因组DNA中PCR扩增Npx和NapA基因，克隆到pCyt3中，分别生成pSVB5和pJP23。通过对克隆DNA的测序，我们证实在克隆过程中没有突变被引入到这两个基因中。IPTG可诱导这两个基因表达，其产物约占细胞总蛋白的2%。两个过表达蛋白的表观分子量与推导出的氨基酸序列预测的分子量具有良好的相关性。两种蛋白均纯化至均匀性，纯化后的蛋白分别用于培养多克隆血清。电感耦合等离子体质谱（ICP-MS）分析表明，没有金属辅助因子与Npx或NapA共纯化。(3)我们将质粒pJP23 （napA）引入到携带ahpC突变的大肠杆菌菌株TA4315中，该突变编码烷基过氧化氢还原酶的一个亚基。当使用IPTG过表达NapA时，携带pJP23的TA4315能够在5iM异丙烯过氧化氢或t-丁基过氧化氢存在下生长。同样，携带pSVB5 （npx）的菌株TA4315在2iM t-丁基过氧化氢激发下能够生长。这些数据表明Npx和NapA具有过氧化物酶或烷基氢过氧化物还原酶的功能。与德克萨斯农工大学微生物学系J. Skare博士合作，分离出了一种高传代、无毒的伯氏疏螺旋体PerR-突变株。初步数据表明，该突变株对5iM过氧化氢具有抗性，而野生型高传代亲本菌株对0.5 iM过氧化氢具有抗性。免疫印迹和PCR分析表明，突变株中perR被破坏，不产生perR。有趣的是，用抗NapA血清检测的免疫印迹显示，NapA（烷基过氧化氢还原酶）在突变菌株中过度表达，部分解释了其过氧化氢抗性表型。(4)此外，我们还鉴定了一个位于napA启动子两侧的假定的PerR靶序列。这些区域与来自亚贻贝芽孢杆菌的氧化抑制结合蛋白PerR的靶序列非常同源。为了证明这些目标序列与PerR之间的相互作用，我们通过PCR扩增了一个251 bp的DNA片段，其中包括napA上游的序列，末端标记为[32P]，并将其作为探针用于与PerR的迁移位移DNA结合试验。在纯化的Per存在下观察到目标序列的迁移性改变，这表明PerR识别目标序列。这些初步结果非常令人鼓舞，因为我们已经证明了PerR在伯氏疏螺旋体基因调控中的潜在作用。摘要：氧化应激部分。我们提出了一个伯氏疏螺旋体对氧化应激反应的模型。因为该系统能促进伯氏疏螺旋体细胞在O2胁迫下的体内存活。-和H2O2，我们对这一过程及其调控方式特别感兴趣。如前所述，我们已经确定了一个基因perR，它可能参与调节伯氏疏螺旋体对氧化应激和金属限制的基因表达。我的研究重点之一是表征在伯氏疏螺旋体中发现的重要调节蛋白，并评估PerR， Npx和NapA在体内生存中的作用。这些研究将有助于更好地了解伯氏疏螺旋体的发病机制。

Since important virulence factors produced by pathogenic bacteria (toxins, adhesins, etc.) are generally regulated by environmental signals (temperature, pH, ion/metal concentration etc.), we have begun to identify these regulatory systems in Borrelia burgdorferi. SDS-PAGE and immunoblotting analysis of proteins from B. burgdorferi growth in modified Barbour-Stoenner-Kelly (BSK-II) media treated with metal chelators such as EDDHA, 2-2 dypiryldyl (DIP), or chelex, suggest that B. burgdorferi alters protein expression in response to decreasing metal concentrations When other bacterial pathogens (Neisseria, E. coli, etc.) were studied using similar experimental approaches, regulatory systems that were identified were based upon metal-dependent repressor proteins that sensed the intracellular concentration of Fe. Initially, we believed this to be the case for B. burgdorferi. However, several experimental results strongly suggest that the Fe requirements of B. burgdorferi are very low or non-existent . These findings and the fact that B. burgdorferi lacks a respiratory chain suggest that metabolism is not generating reactive oxygen species (ROS) during normal cell growth. As important, with no intracellular Fe, there is no Fenton reaction with ROS, particularly H2O2. Therefore, oxidative challenges to B. burgdorferi cells must come primarily from the host in the form of ROS and/or reactive nitrogen species , NOS. To understand how B. burgdorferi biochemical eliminates or reduces ROS (or NOS) and regulates the enzymes involved in this process, we have concentrated on three proteins. We have cloned a gene encoding a putative metal-dependent repressor protein (PerR) from B. burgdorferi and identified two target sequences using a mobility shift DNA-binding assay. One sequence is 60 bp upstream of the start codon of a putative 2 gene operon encoding a glutamate transporter (gltP) and a NADH peroxidase (npx). The other is 57 bp upstream of the start codon of napA, the gene encoding an alkylhydroperoxide reductase. These data indicate that PerR may be involved in regulating an oxidative stress response by B. burgdorferi. A PerR homolog identified from Bacillus subtilis mediates cellular responses to oxidative stress and metal starvation in that bacterium. Clearly, characterizing the role this regulatory protein plays in the survival response of B. burgdorferi and identifying other genes it regulates will contribute greatly to the understanding of Lyme disease. To date, we have done the following:: (1) To begin characterization of this system, perR from B. burgdorferi was cloned into expression vector pKK223-3, induced with IPTG, and the protein was purified from E. coli XL1-Blue MRFʹ. PerR was purified using a heparin-agarose affinity column and used to generate polyclonal sera to the recombinant protein. The antisera detected PerR in cell lysates of B. burgdorferi and has been used to probe the high-passage perR- mutant. (2) The genes for Npx and NapA were amplified by PCR from B. burgdorferi genomic DNA and cloned into pCyt3 to generating pSVB5 and pJP23, respectively. We confirmed that no mutations were introduced into either gene during the cloning procedure by sequencing the cloned DNA. Expression of both genes was inducible with IPTG, and the products of these genes accounted for >2% of the total cell protein. The apparent molecular weights of the two overexpressed proteins correlated well with the molecular weights predicted from the deduced amino acid sequences. Both proteins have been purified to homogeneity and each purified protein was used to raise polyclonal serum. Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) analysis indicated that no metal co-factor co-purified with Npx or NapA. (3) We introduced the plasmid pJP23 (napA) into E. coli strain TA4315 that carried a mutation in the ahpC, encoding one subunit of alkylhydroperoxide reductase. TA4315 harboring pJP23 was able to grow in the presence of 5iM cumene hydroperoxide or t-Butyl hydroperoxide when NapA was overexpressed using IPTG. Similarly, strain TA4315 harboring pSVB5 (npx) was able to grow when challenged with 2iM t-Butyl hydroperoxide. These data suggest that the Npx and NapA function as peroxidases or alkylhydroperoxide reductases. A PerR- mutant of high-passage, avirulent B. burgdorferi has been isolated in collaboration with Dr. J. Skare, Dept. Of Microbiology, Texas A & M University, College Station, TX. Preliminary data suggests that the mutant strain is resistant to 5iM hydrogen peroxide compared to 0.5 iM for the wild-type, high-passage parent strain. Immunoblot and PCR analyses demonstrated that perR had been disrupted and no PerR was produced in the mutant strain. Interestingly, immunoblots probed with anti-NapA serum indicated that NapA (an alkylhydroperoxide reductase) was being over-expressed in the mutant strain partially explaining its peroxide resistant phenotype. (4) In addition, a putative PerR target sequence which flanks the putative promoters of napA were identified. These regions are very homologous to the target sequences of an oxidative repressor binding protein, PerR, from Bacillus subtilus. To demonstrate interactions between these target sequences and PerR, a 251-bp DNA fragment including the sequence upstream of napA was amplified by PCR, end labeled with [32P], and used as a probe in mobility shift DNA-binding assays with PerR. Altered mobility of the target sequence was observed in the presence of purified Per suggesting that PerR recognized the target sequence. These preliminary results are extremely encouraging, as we have demonstrated a potential role for PerR in gene regulation in B. burgdorferi. SUMMARY: Section on oxidate stress. We have proposed a model for the response of B. burgdorferi to oxidative stress. Because this system 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. As previously mentioned, we have identified a gene, perR, that may be involved in regulating gene expression in response to oxidate stress and metal limitation in B. burgdorferi. One focus of my research has been to characterize this important regulatory protein identified in B. burgdorferi and assess the role of PerR, Npx, and NapA in survival in vivo. These studies will lead to a better understanding of the pathogenesis of B. burgdorferi.