Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi.
Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi.
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DOI：
10.1371/journal.pone.0136707
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Seshu J
中科院分区：
文献类型：
--
作者：
Esteve-Gassent MD;Smith TC 2nd;Small CM;Thomas DP;Seshu J
伯氏疏螺旋体是莱姆病的病原体，根据其蜱虫载体或脊椎动物宿主特有的环境信号改变其基因表达。伯氏疏螺旋体携带一个能够控制细胞内超氧化物水平的超氧化物歧化酶基因（sodA）。此前，在莱姆病的C3H/HeN模型中，sodA被证明是伯氏疏螺旋体感染所必需的。我们使用二维电泳（2-DE）和针对羰基化蛋白的抗体进行免疫印迹分析，以确定在内源性超氧化物产生剂甲基紫藻（MV，百草枯）处理后，sodA突变体的可溶性部分与等基因亲本对照菌株相比被不同氧化的靶标。氧化蛋白的HPLC-ESI-MS/MS分析显示，在MV处理的sodA突变体中，糖酵解途径的几个蛋白（BB0057, BB0020, BB0348）的羰基化增加。与亲本菌株相比，在MV处理后，sodA突变体的ATP和NAD/NADH水平降低，这可能归因于糖酵解途径蛋白质氧化水平的增加。此外，在sodA突变体中也观察到伴侣蛋白HtpG （BB0560）和外表面蛋白a （OspA, BBA15）被氧化。免疫印迹分析显示，与对照菌株相比，sodA突变株的外表面蛋白C （OspC）、Decorin结合蛋白A （DbpA）、纤维连接蛋白结合蛋白（BBK32）、RpoS和BosR水平降低。在gp91/phox−⁄−和iNOS缺陷小鼠中都无法恢复活的sodA突变螺旋体，而在感染小鼠的多个组织样本中检测到的螺旋体DNA水平明显低于亲本菌株。综上所述，这些观察结果表明，在莱姆病小鼠模型中，选择性螺旋体决定因素的氧化增加和关键致病相关脂蛋白水平的降低导致sodA突变体的体内缺陷。这项利用sodA突变体的研究，提供了对伯氏疏螺旋体在其宿主中生存至关重要的适应能力的见解。
Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox −⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.