A high-throughput genetic screen identifies previously uncharacterized <i>Borrelia burgdorferi</i> genes important for resistance against reactive oxygen and nitrogen species

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.伯氏疏螺旋体（Borrelia burgdorferi）是人类莱姆病的致病病原体，在蜱媒宿主与脊椎动物储存宿主体内均会暴露于活性氧与活性氮物种（reactive oxygen and nitrogen species，ROS and RNS）之中。伯氏疏螺旋体的经典氧化应激应答基因谱较为有限，提示其或许存在尚未被发现的基因功能，以帮助自身抵御ROS或RNS暴露。本研究借助转座子插入测序（transposon insertion sequencing，Tn-seq）技术，在体外环境中开展无偏筛选，以寻找参与抵御一氧化氮、过氧化氢与叔丁基过氧化氢的基因。筛选实验共鉴定出66个基因，当这些基因发生敲除时，会导致伯氏疏螺旋体对至少一种应激源的敏感性升高。这些基因既包含已被表征的ROS与RNS抗性介导因子，例如核苷酸切除修复通路组分以及核黄素转运蛋白亚基，同时也涵盖了全新的潜在抗性候选基因。在Tn-seq筛选中，DNA修复突变体对RNS的敏感性最为突出，针对单个转座子突变体的生存实验证实，假定的核糖核酸酶BB0839参与了一氧化氮抗性的调控。与之相反，缺失预测内膜蛋白或转运蛋白的突变体对ROS的敏感性最高，通过单个转座子突变体与互补菌株实验，验证了BB0017、BB0164与BB0202这三种膜蛋白在ROS敏感性调控中的作用。进一步分析显示，Tn::bb0164突变体的细胞内锰离子水平显著降低，这揭示了BB0164在伯氏疏螺旋体锰离子稳态调控中的全新功能。利用包含39个转座子突变体的微型文库感染C57BL/6与gp91phox-/-小鼠，结果表明体外筛选所鉴定出的诸多基因，在小鼠体内感染过程中均为必需基因。综上，本研究的数据为伯氏疏螺旋体应对ROS与RNS的机制提供了新的认知，并提示该应答过程与该病原体在宿主体内的定植成功密切相关。