Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
J Bacteriol. 2010 Apr;192(8):2077-84. doi: 10.1128/JB.01324-09. Epub 2010 Feb 19.
Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100 degrees C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.
贝氏考克斯体是一种革兰氏阴性、专性细胞内细菌病原体，存在于宿主细胞内的溶酶体样隔室中，称为寄生空泡。在这项研究中，我们对属于过氧化物酶 2-半胱氨酸亚家族的一种贝氏考克斯体硫醇特异性过氧化物酶进行了表征，通常称为菌铁蛋白共迁移蛋白（BCP）。考克斯氏菌 BCP 最初通过二维西南印迹（SW）印迹技术识别为病原体蛋白质组中的一种潜在 DNA 结合蛋白，并用生物素化的考克斯氏菌基因组 DNA 探测。通过基质辅助激光解吸电离串联飞行时间质谱（MALDI-TOF/TOF MS）证实 DNA 结合蛋白的身份为 BCP（CBU_0963）。生成了重组考克斯氏菌 BCP（rBCP），并通过两种独立的方法证明了其 DNA 结合能力，包括 SW 印迹和电泳迁移率变动分析（EMSA）。rBCP 还在体外显示出需要硫氧还蛋白-硫氧还蛋白还原酶（Trx-TrxR）的过氧化物酶活性。rBCP 的 DNA 结合和过氧化物酶活性在热变性（100°C，10 分钟）后丧失。通过对大肠杆菌 bcp 突变体的反式互补，证明了考克斯氏菌 bcp 的功能表达，这表现在该菌株能够在含有叔丁基过氧化氢的氧化应激生长培养基中生长，其水平与野生型亲本菌株相当，甚至显著高于野生型亲本菌株，显著高于 bcp 突变体水平（P <0.05）。rBCP 还被发现能够保护超螺旋质粒 DNA 免受体外氧化损伤（即切口）。bcp 基因的最大表达与上皮（Vero）宿主细胞系同步感染实验中病原体的早期（第 2 至 3 天）指数生长阶段一致。总的来说，这些结果表明，考克斯氏菌 BCP 结合 DNA，并可能在细胞内复制过程中通过代谢产生的内源性过氧化物副产物来解毒。
