Identification of a Novel Small RNA Modulating <em>Francisella tularensis</em> Pathogenicity

Francisella tularensis is a highly virulent bacterium responsible for the zoonotic disease tularemia. It is a facultative intracellular pathogen that replicates in the cytoplasm of host cells, particularly in macrophages. Here we show that F. tularensis live vaccine strain (LVS) expresses a novel small RNA (sRNA), which modulates the virulence capacities of the bacterium. When this sRNA, designated FtrC (for Francisella tularensisRNA C), is expressed at high levels, F. tularensis replicates in macrophages less efficiently than the wild-type parent strain. Similarly, high expression of FtrC reduces the number of viable bacteria recovered from the spleen and liver of infected mice. Our data demonstrate that expression of gene FTL_1293 is regulated by FtrC. Furthermore, we show by in vitro gel shift assays that FtrC interacts specifically with FTL_1293 mRNA and that this happens independently of the RNA chaperone Hfq. Remarkably, FtrC interacts only with full-length FTL_1293 mRNA. These results, combined with a bioinformatic analysis, indicate that FtrC interacts with the central region of the mRNA and hence does not act by sterically hindering access of the ribosome to the mRNA. We further show that gene FTL_1293 is not required for F. tularensis virulence in vitro or in vivo, which indicates that another unidentified FtrC target modulates the virulence capacity of the bacterium.土拉弗朗西斯菌（Francisella tularensis）是一种高致病性细菌，可引发人畜共患病土拉菌病。它是一类兼性胞内寄生菌，能够在宿主细胞的细胞质中增殖，尤其偏好巨噬细胞。本研究显示，土拉弗朗西斯菌活疫苗株（live vaccine strain, LVS）可表达一种新型小RNA（small RNA, sRNA），该RNA可调控该细菌的毒力特性。当这种被命名为FtrC（即Francisella tularensis RNA C）的小RNA高表达时，土拉弗朗西斯菌在巨噬细胞内的增殖效率相较于野生型亲本菌株显著降低。类似地，FtrC的高表达还会减少从感染小鼠脾脏与肝脏中回收的活菌数量。本研究数据证实，基因FTL_1293的表达受FtrC调控。此外，通过体外凝胶迁移实验（in vitro gel shift assays）我们发现，FtrC可与FTL_1293 mRNA特异性结合，且这一结合过程不依赖RNA分子伴侣Hfq。值得注意的是，FtrC仅能与全长FTL_1293 mRNA结合。结合生物信息学分析（bioinformatic analysis）结果，上述结果表明FtrC可与该mRNA的中央区域结合，因此并非通过空间位阻阻碍核糖体与mRNA的结合来发挥作用。我们进一步证实，无论在体外还是体内环境中，基因FTL_1293都不是土拉弗朗西斯菌毒力所必需的，这意味着存在另一种尚未被鉴定的FtrC靶标来调控该细菌的毒力能力。