Ribonucleotide Reductases of <em>Salmonella</em> Typhimurium: Transcriptional Regulation and Differential Role in Pathogenesis

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses.核糖核苷酸还原酶（Ribonucleotide reductases, RNRs）是一类通过还原核糖核苷酸实现脱氧核糖核苷酸从头合成的必需酶。目前已发现三类RNR（I、II、III型），各类别具有不同的氧依赖性与生化特性。肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium, S. Typhimurium）的基因组中携带有Ia型、Ib型及III型RNR编码基因。本研究针对鼠伤寒沙门氏菌中三类RNR的转录调控机制，及其在巨噬细胞与上皮细胞感染过程中的差异功能展开了探究。通过对两种主要转录调节因子NrdR与Fur的双缺失突变分析，本研究证实Fur可特异性阻遏Ib型RNR的表达，而NrdR则作为三类RNR的全局性阻遏因子。此外，本研究通过电泳迁移率变动分析（electrophoretic mobility shift assays, EMSA）鉴定并验证了nrdHIEF启动子区域内的Fur识别序列。为阐明各类RNR在感染过程中的具体作用，本研究构建了鼠伤寒沙门氏菌的单、双RNR突变株（同时包含Fur与NrdR突变株），并利用巨噬细胞及上皮细胞系开展感染实验。实验结果表明，Ia型RNR主要负责细菌入侵宿主后，在巨噬细胞与上皮细胞内增殖过程中的脱氧核糖核苷酸合成。在此培养条件下，Ib型与III型RNR似乎均非细菌生长所必需。但在巨噬细胞感染的最初数小时内，Ib型RNR可在nrdAB突变株中维持一定的生长水平。本研究结果提示，在巨噬细胞感染早期，宿主氧化爆发所产生的过氧化氢与DNA损伤可通过NrdR与Fur依赖的nrdHIEF去阻遏通路激活Ib型RNR，进而促进脱氧核糖核苷酸合成，帮助细菌抵御宿主防御系统。