Characterization of the oxidative stress regulon of Campylobacter jejuni

Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.

研究背景：空肠弯曲杆菌（Campylobacter jejuni, C. jejuni）作为肠道致病菌，在肠道定植过程中必须抵御由自身代谢、宿主免疫系统及肠道菌群产生的活性氧（reactive oxygen species, ROS）的毒性作用。阐明空肠弯曲杆菌的氧化应激防御机制，对于理解弯曲杆菌的致病生理过程至关重要。
研究结果：本研究通过转录组分析（transcriptional profiling）、基因诱变及表型分析，对空肠弯曲杆菌的氧化应激防御机制进行了系统解析。结果显示，空肠弯曲杆菌在暴露于过氧化氢（hydrogen peroxide, H₂O₂）、异丙苯过氧化氢（cumene hydroperoxide, CHP）或甲萘醌（menadione, MND）后，其转录组变化具有氧化剂特异性，且涉及多种生物学通路相关基因的差异表达——涵盖经典氧化应激防御系统、热休克应答、DNA修复与代谢、脂肪酸及荚膜生物合成、多药外排泵等通路。为明确过氧化物感应调节因子PerR，本研究构建了同基因缺失突变株，并将其转录组谱与野生型菌株进行对比，最终鉴定出66个属于PerR调节子（PerR regulon）的基因。PerR可通过铁和/或过氧化氢依赖及非依赖两种方式调控基因表达。perR突变株的运动能力受损，且在鸡肠道定植模型中致病性减弱。对超氧化物歧化酶（superoxide dismutase, SodB）、烷基过氧化氢酶（alkyl-hydroxyperoxidase, AhpC）及过氧化氢酶（catalase, KatA）开展的诱变与表型研究证实，这些蛋白在氧化应激防御及鸡肠道定植过程中发挥关键作用。
研究结论：本研究揭示了PerR、铁代谢与氧化应激防御之间的相互调控关系，并阐明了三者在空肠弯曲杆菌于鸡盲肠内定植和/或存活过程中的重要作用。
关键词：针对3种氧化剂（H₂O₂、甲萘醌及异丙苯过氧化氢）的转录应答，以及perR调节子的表征（野生型与perR突变株的转录组对比分析）。
为探究空肠弯曲杆菌对氧化剂暴露的转录应答，本研究向50 mL菌液中分别添加终浓度为1 mM的H₂O₂、CHP或亚硫酸氢钠甲萘醌（MND）。以添加等量无菌水或二甲基亚砜（dimethyl sulfoxide, DMSO）的细菌培养物作为对照样本，用于对应氧化剂处理下的转录谱分析。此外，为探究过量铁存在时空肠弯曲杆菌对H₂O₂暴露的转录应答，本研究在H₂O₂处理前15 min，向菌液中添加终浓度为40 μM的硫酸亚铁。于氧化剂添加10 min后，提取总RNA并进行微阵列杂交（microarray hybridization）检测。为鉴定PerR调节子，本研究将空肠弯曲杆菌野生型菌株NCTC 11168及perR突变株接种于含250 mL MEMα培养基（MEMα medium）的500 mL培养瓶中培养。至对数中期时，取50 mL菌液转移至100 mL培养瓶中，分别添加硫酸亚铁和/或H₂O₂。于H₂O₂添加10 min后收集菌体，提取总RNA。