The Extraintestinal Pathogenic Escherichia coli Factor RqlI Constrains the Genotoxic Effects of the RecQ-Like Helicase RqlH.
The Extraintestinal Pathogenic Escherichia coli Factor RqlI Constrains the Genotoxic Effects of the RecQ-Like Helicase RqlH.
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DOI：
10.1371/journal.ppat.1005317
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发表时间：
2015-12
期刊：
影响因子：
6.7
通讯作者：
Mulvey MA
中科院分区：
文献类型：
--
作者：
Russell CW;Mulvey MA
肠道外致病性大肠杆菌定植于人体肠道，并可扩散到身体其他部位，诱发尿路感染、败血症和脑膜炎等疾病。大肠杆菌固有的遗传多样性和大量未研究的基因阻碍了对感染过程的全面了解。在这里，我们专注于未表征的基因rqlI，我们的实验室最近在斑马鱼感染模型所需的细菌基因的n-seq筛选中发现了它。我们证明ΔrqlI突变体在低氧条件下经历生长缺陷和增加的DNA胁迫。在Δrql菌株抑制基因突变的遗传筛选中，我们发现Δrql突变体的缺陷可归因于RqlH的活性，在其他细菌中，RqlH是RecQ家族的一种解旋酶，含有磷酸核糖基转移酶（PRTase）结构域。在体内和体外实验中，破坏rqlH可挽救ΔrqlI菌株，而单独表达rqlH可激活与细菌丝化一致的SOS反应，提高对DNA损伤的敏感性，并增加突变率。截断突变体分析表明，在缺乏RqlI的情况下，RqlH的毒性是由于其PRTase结构域。补充研究表明，RqlH的毒性是通过RqlI中的重叠结构域以上下文依赖的方式调节的。考虑到这两种蛋白质在物理上相互作用并形成一个操纵子，这种调节似乎是直接的。有趣的是，RqlH和RqlI同源物是由不同的细菌群编码的，但在许多这些微生物中，特别是在革兰氏阳性生物体中，RqlH是在RqlI缺失的情况下发现的。总之，这项工作表明RqlH和RqlI可以以一种菌株特异性的方式起作用，类似于毒素-抗毒素系统，其中毒性是由非典型解旋酶相关的PRTase结构域介导的。肠外致病性大肠杆菌（expc）引起大多数尿路感染，也能够感染人类宿主的血液、脑膜和其他各种部位。这些感染正变得越来越难以治疗，因为exp菌株对临床上常用的许多抗生素产生了耐药性。改进治疗策略的发展需要更深入地了解促进宿主内ExPEC适应性和毒力的因素。在遗传筛选中，我们发现了一种功能未知的蛋白RqlI，它可以促进expc在不同宿主环境中的存活。我们发现RqlI与RqlH结合并协同工作，RqlH是一种在其他细菌中被证明可以解开DNA的蛋白质。在缺乏RqlI的情况下，我们发现RqlH会对ExPEC产生毒性，导致DNA损伤和生长减慢。RqlH的一个特定部分被预测会操纵构成DNA的核苷酸，这是造成这种毒性的原因。RqlH在不受RqlI控制的情况下能够抑制细菌生长，这表明RqlI的特异性失活可能在对抗ExPEC和其他表达这些蛋白的病原体方面具有治疗价值。
Extraintestinal pathogenic Escherichia coli colonize the human gut and can spread to other body sites to induce diseases such as urinary tract infections, sepsis, and meningitis. A complete understanding of the infection process is hindered by both the inherent genetic diversity of E. coli and the large number of unstudied genes. Here, we focus on the uncharacterized gene rqlI, which our lab recently uncovered in a Tn-seq screen for bacterial genes required within a zebrafish model of infection. We demonstrate that the ΔrqlI mutant experiences a growth defect and increased DNA stress in low oxygen conditions. In a genetic screen for suppressor mutations in the Δrql strain, we found that the shortcomings of the Δrql mutant are attributable to the activity of RqlH, which is known in other bacteria to be a helicase of the RecQ family that contains a phosphoribosyltransferase (PRTase) domain. Disruption of rqlH rescues the ΔrqlI strain in both in vivo and in vitro assays, while the expression of RqlH alone activates the SOS response coincident with bacterial filamentation, heightened sensitivity to DNA damage, and an increased mutation rate. The analysis of truncation mutants indicates that, in the absence of RqlI, RqlH toxicity is due to its PRTase domain. Complementary studies demonstrate that the toxicity of RqlH is modulated in a context-dependent fashion by overlapping domains within RqlI. This regulation is seemingly direct, given that the two proteins physically interact and form an operon. Interestingly, RqlH and RqlI orthologs are encoded by a diverse group of bacteria, but in many of these microbes, and especially in Gram-positive organisms, rqlH is found in the absence of rqlI. In total, this work shows that RqlH and RqlI can act in a strain-specific fashion akin to a toxin-antitoxin system in which toxicity is mediated by an atypical helicase-associated PRTase domain. Extraintestinal pathogenic Escherichia coli (ExPEC) cause the majority of urinary tract infections, and are also able to infect the bloodstream, meninges, and various other sites within the human host. These infections are becoming increasingly difficult to treat as ExPEC strains gain resistance to many of the antibiotics that are commonly used in the clinic. The development of improved treatment strategies requires a deeper understanding of the factors that promote ExPEC fitness and virulence within the host. In genetic screens, we identified a functionally uncharacterized protein, RqlI, which promotes ExPEC survival within diverse host environments. We find that RqlI binds to and works in tandem with RqlH, a protein that has been shown in other bacteria to unwind DNA. In the absence of RqlI, we found that RqlH can become toxic to ExPEC, causing DNA damage and slower growth. A specific part of RqlH that is predicted to manipulate the nucleotides that make up DNA is responsible for this toxicity. The ability of RqlH to inhibit bacterial growth when not held in check by RqlI suggests that the specific inactivation of RqlI could have therapeutic value in combating ExPEC and other pathogens that express these proteins.