The Oxidative Stress Response In B. burgdorferi

伯氏疏螺旋体的氧化应激反应

• 批准号: 6987024
• 负责人: Frank Gherardini
• 金额: --
• 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/6987024
• 关键词: Borrelia; DNA binding protein; DNA footprinting; Lyme disease; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; biological signal transduction; free radical oxygen; gene expression; genetic regulation; genetic regulatory element; high performance liquid chromatography; host organism interaction; intermolecular interaction; nitric oxide; oxidative stress; pathologic process; peroxidases; protein structure function; regulatory gene; superoxide dismutase; western blottings

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 biochemically 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 (BB0647) from B. burgdorferi and identified two target sequences using a mobility shift DNA-binding assay. One sequence is 130 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 130 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)Stress-related genes appear to be regulated by a putative metal-dependent DNA binding protein (BB0647) that has 50.7% similarity to the peroxide-specific stress response repressor of Bacillus subtilis, PerR. We overexpressed and purified this protein from E. coli and designated it Borrelia oxidative stress regulator, BosR. BosR bound to a 50-nt region 180 bp upstream of the napA transcriptional start site and required DTT and Zn2+ for optimum binding. Unlike the B. subtilis PerR repressor, Fe2+ and Mn2+ were not required for binding, and oxidizing agents, such as t-butyl peroxide, enhanced, not eliminated, BosR binding to the napA promoter region. Surprisingly, transcriptional fusion analysis indicated that BosR exerted a positive regulatory effect on napA that is inducible with t-butyl peroxide. Based on these data, we propose that, despite the similarity to PerR, BosR functions primarily as a transcriptional activator and not a repressor of oxidative stress response in B. burgdorferi. (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 BosR- 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 5 mM hydrogen peroxide compared to 0.5 mM for the wild-type, high-passage parent strain. Immunoblot and PCR analyses demonstrated that perR had been disrupted and no BosR 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. 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.

由于致病细菌产生的重要毒力因子（毒素，粘附素等）通常受环境信号（温度，pH，离子/金属浓度等）的调节，因此我们已经开始识别Borlelia burgdorferi中的这些调节系统。经过修饰的Bargdorferi生长中的蛋白质的SDS-PAGE和免疫印迹分析在经过改良的Barbour-Stoenner-Kelly（BSK-II）中，用金属螯合剂（例如Eddha，2-2 dipiryldyl（DIP）或CHELEX）处理的培养基（BSK-II）培养基，表明B. burgdorferi Allgdorferi Allgdorferi Allgdorferi Allgdorferi All Grgdorferi All Grgdorferi s ant prots prot inters prot inters prot s ant prot s Alters prot in vations venters sandersions降低了金属浓度。当使用类似的实验方法研究其他细菌病原体（奈瑟菌，大肠杆菌等）时，鉴定出的调​​节系统是基于感觉到Fe细胞内浓度的金属依赖性抑制剂蛋白。最初，我们认为B. Burgdorferi是这种情况。但是，几个实验结果强烈表明，伯氏芽孢杆菌的Fe要求非常低或不存在。这些发现以及B. burgdorferi缺乏呼吸链的事实表明，在正常细胞生长期间，代谢并未产生活性氧（ROS）。同样重要的是，没有细胞内Fe，与ROS，尤其是H2O2没有芬顿反应。因此，对B. burgdorferi细胞的氧化挑战必须主要来自宿主，以ROS和/或反应性氮种NOS的形式来自宿主。为了了解B. burgdorferi生物化学如何消除或减少ROS（或NOS）并调节参与此过程的酶，我们集中于三种蛋白质。我们已经克隆了一个基因，该基因编码了B. burgdorferi的推定金属依赖性抑制剂蛋白（BB0647），并使用迁移率偏移DNA结合测定法鉴定了两个靶序列。一个序列是编码谷氨酸转运蛋白（GLTP）和NADH过氧化物酶（NPX）的假定2基因操纵子的起始密码子上游的130 bp。另一个是NAPA起始密码子上游的130 bp，该基因编码了烷基氢氧化烷基还原酶。这些数据表明，PER可能参与了B. burgdorferi的氧化应激反应。从枯草芽孢杆菌中鉴定出的Perr同源物介导了该细菌中对氧化应激和金属饥饿的反应。显然，表征该调节蛋白在汉堡芽孢杆菌的生存反应中所起的作用，并确定其调节的其他基因将对莱姆病的理解产生巨大贡献。迄今为止，我们已经完成了以下操作：（1）与应力相关的基因似乎受推定的金属依赖性DNA结合蛋白（BB0647）的调节，该蛋白与枯草芽孢杆菌的过氧化物特异性应激响应抑制剂具有50.7％的相似性。我们从大肠杆菌中过表达并纯化了该蛋白质，并指定了伯罗氏氧化应激调节剂BOSR。 BOSR与NAPA转录起始位点上游的50-NT区域结合，并需要DTT和Zn2+才能获得最佳结合。与枯草芽孢杆菌Perr抑制剂不同，结合不需要Fe2+和Mn2+，而氧化剂（例如T-丁基过氧化氢），增强，未消除，BOSR与NAPA启动子区域的结合。出乎意料的是，转录融合分析表明，BOSR对NAPA产生了积极的调节作用，该作用与丁基过氧化叔丁基诱导。基于这些数据，我们建议，尽管与PER相似，但BOSR主要起作用作为转录激活剂，而不是B. burgdorferi中氧化应激反应的阻遏物。 （2）NPX和NAPA的基因通过B. burgdorferi基因组DNA的PCR扩增，并分别克隆到PCYT3中，以生成PSVB5和PJP23。我们证实，在克隆过程中，通过测序克隆的DNA在克隆过程中未引入任何突变。这两个基因的表达均与IPTG诱导，这些基因的产物占总细胞蛋白的2％。两种过表达蛋白的表观分子量与推导的氨基酸序列预测的分子量很好地相关。两种蛋白质均已纯化为均匀性，并使用每种纯化的蛋白质来提高多克隆血清。电感耦合的等离子体质量光谱（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）引入了大肠杆菌菌株TA4315中，该大肠杆菌菌株在AHPC中携带突变，编码一个烷基羟基氧化烷氧化烷氧化物还原酶的一个亚基。当使用IPTG过表达NAPA时，携带PJP23的TA4315能够在5IM液过氧化氢或T叔丁基氢过氧化物的存在下生长。同样，携带PSVB5（NPX）的菌株TA4315在用2IM T丁基氢过氧化物挑战时能够生长。这些数据表明，NPX和NAPA充当过氧化物酶或烷基氢氧化烷还原酶。 Aviruity B. Burgdorferi的一个高通的爆发物与德克萨斯州A＆M University的Microbiology系J. Skare博士合作隔离。初步数据表明，对于野生型，高通量母体菌株而言，突变菌株对过氧化5 mM的过氧化氢具有抗性。免疫印迹和PCR分析表明，Perr已被破坏，在突变菌株中没有产生BOSR。有趣的是，用抗NAPA血清探测的免疫印迹表明，NAPA（一种烷基氢化氧化物还原酶）在突变菌株中被过表达，部分解释了其过氧化抗氧化抗氧化物的耐药性表型。摘要：有关牛应力的部分。我们提出了一个模型，用于墨芽孢杆菌对氧化应激的响应。由于该系统将在受宿主细胞的O2.-和H2O2挑战时促进伯氏芽孢杆菌细胞的体内存活率，因此我们对这一过程及其调节方式特别感兴趣。如前所述，我们已经确定了一个基因Perr，该基因可能参与调节基因表达，以响应牛B. b. bugdorferi中的压力和金属限制。我的研究重点是表征在B. burgdorferi中鉴定出的这种重要调节蛋白，并评估PERR，NPX和NAPA在体内生存中的作用。这些研究将使对B. burgdorferi的发病机理有更好的了解。