CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi
CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi
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DOI：
10.1128/iai.00882-10
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发表时间：
2011-02-01
影响因子：
3.1
通讯作者：
Seshu, J.
中科院分区：
文献类型：
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作者：
Karna, S. L. Rajasekhar;Sanjuan, Eva;Seshu, J.
碳储存调节因子 A (CsrA) 是一种 RNA 结合蛋白，在许多细菌物种中都具有通过调节多种代谢过程发挥核心调节作用的特征。我们最近发现，伯氏疏螺旋体 (Borrelia burgdorferi) 中的 CsrA 同源物 (CsrA(Bb)，BB0184) 在哺乳动物宿主特定条件下响应伯氏疏螺旋体的繁殖而上调。为了进一步描述 CsrA(Bb) 的作用，我们在伯氏疏螺旋体菌株 B31 (ML23) 的线性质粒 25 阴性分离株中产生了命名为 ES10 的缺失突变体。通过PCR和Southern印迹杂交筛选缺失突变体，并通过免疫印迹分析证实ES10中CsrA(Bb)合成的缺乏。对在 pH 6.8/37 摄氏度下繁殖的 ES10 的分析表明，与在这些条件下繁殖的亲本野生型菌株相比，OspC、DbpA、BBK32 和 BBA64 的水平显着降低，而 OspA 或 P66 的水平没有显着变化。此外，ES10 中两种调节蛋白 RpoS 和 BosR 的水平也低于对照菌株。对从亲本菌株和 csrA(Bb) 突变体中提取的总 RNA 进行定量实时逆转录 PCR 分析显示，基因表达存在显着差异，与蛋白质水平的变化一致。在以每只小鼠 103 或 10(5) 个螺旋体对 C3H/HeN 小鼠进行皮内针接种后，csrA(Bb) 突变体和反式互补菌株均不能感染。 CsrA(Bb) 介导的调节分子基础的进一步表征将为了解伯氏疏螺旋体的病理生理学提供重要的见解。
Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrA(Bb), BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrA(Bb), we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrA(Bb) in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37 degrees C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrA(Bb) mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrA(Bb) mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 103 or 10(5) spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrA(Bb) will provide significant insights into the pathophysiology of B. burgdorferi.